Compositions and methods for synthesizing 5′-Capped RNAs

ABSTRACT

Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form m7Gppp[N2′Ome]n[N]m wherein m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.15/761,957, filed Mar. 21, 2018, which is a national phase applicationunder 35 U.S.C. § 371 of International application No.PCT/US2016/052670, filed Sep. 20, 2016, which claims priority from U.S.Provisional Application No. 62/221,248, filed Sep. 21, 2015, thecontents of which are hereby incorporated by reference herein in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 19, 2018, isnamed 095109-000520US-1105435_SL.txt and is 823 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for thesynthesis of 5′Capped RNAs. In particular aspects, the present inventionrelates to novel Cap containing initiating oligonucleotide primershaving natural or modified 5′-Cap 0, Cap 1, Cap 2 ortrimethylguanosine-Cap (TMG-Cap) structures. In additional aspects, thepresent invention relates to methods for efficiently generating andusing the same for preparing 5′-Capped RNAs are provided.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art in the present invention.

Messenger RNA (mRNA), encoding physiologically important proteins fortherapeutic applications, has shown significant advantages overDNA-based plasmid and viral vectors for delivering genetic material. Themost important of these advantages are:

-   -   (i) high level of safety (reduces potential genome damage from        viral or plasmid integration),    -   (ii) mRNA delivery results in immediate protein expression        (unlike delayed responses that generally occur with plasmids),    -   (iii) mRNA allows for robust dose-dependent control over        expression of proteins and    -   (iv) the simplicity of large scale synthesis of mRNAs compared        to manufacturing of plasmid and viral vectors.

Messenger RNAs can be encoded for virtually any known protein and can bedelivered to specific tissues and organs by a variety of methods knownto those skilled in the art. Once delivered, these mRNAs directribosomal protein expression within targeted tissues resulting in theproduction of many hundreds of proteins per mRNA molecule.

Several structural elements, present in each active mRNA molecule, areutilized to translate the encoded proteins efficiently. One of theseelements is a Cap structure on the 5′-end of mRNAs, which is present inall eukaryotic organisms (and some viruses). Naturally occurring Capstructures comprise a ribo-guanosine residue that is methylated atposition N7 of the guanine base. This 7-methylguanosine (^(7m)G) islinked via a 5′- to 5′-triphosphate chain at the 5′-end of the mRNAmolecule. Throughout this application, 7m and m7 are usedinterchangeably with equivalent meaning. The presence of the ^(7m)Gpppfragment on the 5′-end is essential for mRNA maturation, it:

-   -   protects the mRNAs from degradation by exonucleases,    -   facilitates transport of mRNAs from the nucleus to the cytoplasm        and    -   plays a key role in assembly of the translation initiation        complex (Cell 9:645-653, (1976); Nature 266:235, (1977);        Federation of Experimental Biologists Society Letter 96:1-11,        (1978); Cell 40:223-24, (1985); Prog. Nuc. Acid Res. 35:173-207,        (1988); Ann. Rev. Biochem. 68:913-963, (1999); J. Biol. Chem.        274:30337-3040, (1999)).

Only those mRNAs that carry the Cap structure are active in Capdependent translation; “decapitation” of mRNA results in an almostcomplete loss of their template activity for protein synthesis (Nature,255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc.Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

Another element of eukaryotic mRNA is the presence of 2′-O-methylnucleoside residues at transcript position 1 (Cap 1), and in some cases,at transcript positions 1 and 2 (Cap 2). The 2′-O-methylation of mRNA isrequired for higher efficacy of mRNA translation in vivo (Proc. Natl.Acad. Sci. USA, 77:3952-3956 (1980)) and further improves nucleasestability of the 5′-capped mRNA. The mRNA with Cap 1 (and Cap 2) is adistinctive mark that allows cells to recognize the bona fide mRNA 5′end, and in some instances, to discriminate against transcriptsemanating from infectious genetic elements (Nucleic Acid Research 43:482-492 (2015)).

A primary mRNA transcript carries a 5′-triphosphate group (5′-pppmRNA)resulting from initiation of RNA synthesis starting with NTP (typicallyGTP) in vivo. Conversion of 5′-triphosphorylated end of mRNA transcriptinto a Cap structure (Cap 0) occurs via several enzymatic steps (J.Biol. Chem. 250:9322, (1975); J. Biol. Chem. 271:11936, (1996); J. Biol.Chem. 267:16430, (1992)). These enzymatic reactions steps include:

-   -   Step 1: RNA triphosphatase converts 5′-triphosphate of mRNA to a        5′-diphosphate, pppN₁(pN)_(x)→ppN₁(pN)_(x)+ inorganic phosphate;    -   Step 2: RNA guanyltransferase uses GTP to transfer a GMP residue        to the 5′-diphosphate of the mRNA,        ppN₁(pN)_(x)+GTP→G(5′)ppp(5′)N₁(pN)_(x)+ inorganic        pyrophosphate; and    -   Step 3: guanine-7-methyltransferase, uses S-adenosyl-methionine        (AdoMet) as a cofactor and transfers the methyl group from        AdoMet to the 7-nitrogen of the guanine base,        G(5′)ppp(5′)N₁(pN)_(x)+ AdoMet→^(7m)G(5′)ppp(5′)N₁(pN)_(x)+        AdoHyc).        The RNA that results from these enzymatic activities is referred        to as “5′ capped RNA” or “capped RNA”, and the combination of        enzymes involved in this process that results in formation of        “capped RNA” are referred to as “capping enzymes”. Capping        enzymes, including cloned forms of such enzymes, have been        identified and purified from many sources and are well known in        the art (Prog. Nucleic Acid Res. Mol. Biol. 66:1-40, (2001);        Prog. Nucleic Acid Res. Mol. Biol. 50:101-129, (1995); and        Microbiol. Rev. 44:175, (1980)). The capped RNA that results        from the addition of the cap nucleotide to the 5′-end of primary        RNA by capping enzymes has been referred to as capped RNA having        a “Cap 0 structure” (J. Biol. Chem. 269:14974-14981, (1994); J.        Biol. Chem. 271:11936-11944, (1996)). Capping enzymes have been        used to synthesize capped RNA having a Cap 0 structure in vitro        (J. Biol. Chem. 255:11588, (1980); Proc. Natl. Acad. Sci. USA        94:9573, (1997); J. Biol. Chem. 267:16430, (1992); J. Biol.        Chem. 269:14974, (1994); and J. Biol. Chem. 271:11936, (1996)).

Capped RNA having a 5′-Cap 0 structure can be further transformed invivo to a “Cap 1” structure by the action of (nucleoside-2′-O—)methyltransferase (J. Biol. Chem. 269:14974-14981, (1994); J. Biol.Chem. 271:11936-11944, (1996); and EMBO 21:2757-2768, (2002)). Forexample, vaccinia mRNA (nucleoside-2′-O) methyltransferase can catalyzemethylation of the 2′-hydroxyl group of the 5′-penultimate nucleotide of5′-capped RNA having a Cap 0 structure by the following reaction:^(7m)G(5′)ppp(5′)N₁pN₂(pN)_(x)+AdoMet→^(7m)G(5′)ppp(5′)N₁^(2′-OMe)pN2(pN)_(x)+AdoHyc.

Dimethylated capped RNAs having a Cap 1 structure have been reported tobe translated more efficiently than capped RNAs having a Cap 0 structure(Nucleic Acids Res. 26:3208, (1998)). Eukaryotic cells utilize another(nucleoside-2′-O) methyltransferase (for example hMTR2 in human cells(Nucleic Acids Res. 39:4756 (2011)) to catalyze methylation of the2′-hydroxyl group of the second transcribed nucleotide of 5′-capped RNAto convert the Cap 1 structure to a Cap 2 structure by the followingreaction:^(7m)G(5′)ppp(5′)N₁ ^(2′-OMe)pN₂(pN)_(x)+AdoMet→^(7m)G(5′)ppp(5′)N₁^(2′-OMe)pN₂ ^(2′-OMe)(pN)_(x)+AdoHyc.Approximately 50% of eukaryotic mRNAs have a Cap 2 structure.

In order to produce long functional RNAs for various biological studies,a method of in vitro enzymatic synthesis of primary RNA was developed inthe mid-1980s (Methods Enzymol. 180:51-62, (1989); Nucl. Acids Res.,10:6353-6362, (1982); Meth. Enzymol., 180:42-50 (1989); Nucl. AcidsRes., 12:7035-7056, (1984) and Nucleic Acid Research 15: 8783-8798,(1987)).

After in vitro transcription, the primary mRNA transcript carrying5′-triphosphate group can be further capped with capping enzymes.However, in vitro enzymatic 5′-capping is expensive, laborious,inefficient and difficult to control.

In view of these disadvantages, another method was developed for the invitro synthesis of capped mRNA where a chemically synthesizeddinucleotide ^(7m)G(5′)ppp(5′)G (also referred to as mCAP) is used toinitiate transcription (RNA 1: 957-967, (1995)). The mCAP dinucleotidecontains the 5′-Cap 0 structure of mature mRNA but does not have2′-O-methyl nucleosides characteristic for Cap 1 and Cap 2 structures.

However, there are two main disadvantages attributed to initiation of invitro transcription using synthetic mCAP dinucleotide. The first is astrong competition of mCAP and pppG for initiation of mRNA synthesis.When mRNA is initiated with pppG the resultant ppp-mRNA is inactive intranslation and immunogenic due to the presence of the 5′-triphosphate.Correspondingly, when mRNA is initiated with mCAP the resultant5′-capped-mRNA is active in translation and is not as immunogenic.

In order to improve the ratio of 5′-capped to 5′-uncapped (or5′-triphosphorylated; pppmRNA) mRNAs, an excess of ^(7m)GpppG over pppG(from 4:1 to 10:1) must be used to favor the production of the 5′-Capstructure mRNA transcripts (up to 80-90%). The negative side of thisapproach is a significant reduction of overall yield of mRNA due to afast depletion of GTP supply during transcription and a requirement forlarge quantities of a synthetic mCAP dimer which can be expensive. Aftertranscription an additional treatment of crude mixture, containing both5′-capped mRNA and 5′-pppmRNA, with alkaline phosphatase is necessary toremove uncapped 5′-triphosphate groups from pppmRNA in order to reduceimmunogenicity of synthesized mRNA. The uncapped 5′-OH form of mRNAobtained after phosphatase treatment is inactive and does notparticipate in translation process.

Another disadvantage, a bi-directional initiation, can arise when usinga non-symmetrical mCAP dinucleotide. There is a tendency of the3′-hydroxyl group of either the G or ^(7m)G moiety of ^(7m)GpppG toserve as initiation point for transcriptional elongation with a nearlyequal probability. It typically leads to a synthesis of two isomericRNAs of the form ^(7m)G(5′)pppG(pN)_(n) and G(5′)ppp^(7m)G(pN)_(n), inapproximately equal proportions, depending on conditions of thetranscription reaction (RNA 1: 957-967, (1995)).

To eliminate bi-directional initiation of mRNA synthesis with mCAPdinucleotide a novel modified mCAP analog in which the 3′-OH group of^(7m)G residue is replaced with OCH₃ (“OMe”):^(7m)G(3′-O-Me)pppG (alsoknown as Anti-Reverse Cap Analog (ARCA)) was developed. ARCA initiatesmRNA synthesis only in the correct forward orientation (RNA 7:1486-1495(2001)). Several types of ARCA analogs are known in the art (see, forexample, U.S. Pat. No. 7,074,596). However, a large molar excess of ARCAover pppG is still required to ensure that most mRNA transcriptmolecules have the 5′-Cap structure. A further disadvantage is that anmRNA with a Cap 1 structure cannot be synthesized using a^(7m)GpppG^(2′-OMe) Cap dimer (RNA 1: 957, (1995)) or its ARCA analog.

Presently, the known routes to production of active long mRNAscontaining a Cap 1 structure consist of enzymatic capping and enzymatic2′ O-methylation of the 5′-triphosphorylated mRNA transcript orenzymatic 2′-O-methylation of mCAP-capped or ARCA-capped mRNA precursor(Nucleosides, Nucleotides, and Nucleic Acids, 25:337-340, (2006) andNucleosides, Nucleotides, and Nucleic Acids 25(3):307-14, (2006)). Bothapproaches are quite laborious, difficult to control and, even with asubstantial optimization, neither approach can guarantee a high yield ofa capped and methylated mRNA precursor (J. Gen. Virol., 91:112-121,(2010)). Further, methods for preparing mRNAs with a Cap 2 structure areeven more difficult and results are less predictable. Enzymes forconverting Cap 1 to Cap 2 are not currently commercially available.

Another significant complication of in vitro synthesis of mRNAs,especially in large scale manufacturing, is the necessity to isolate andpurify the active mRNA molecules carrying Cap from all uncapped mRNAforms which are inactive and in some cases immunogenic. Unfortunately,these methods are not trivial and often require a synthesis of modifiedmCAP analogs with conjugated affinity tag moieties allowing for easierisolation and purification of capped RNA transcript. Methods ofsynthesizing mCAP analogs with affinity tags as a reporter/affinitymoiety and novel protocols for isolation of capped RNA from thetranscription reaction mixture are known in the art (see, for example,U.S. Pat. No. 8,344,118). While these approaches are efficient, theyrequire use of more expensive mCAP analogs and they allow forpreparation and isolation of mRNAs containing the Cap 0 structure only.

The in vitro synthesis of natural and modified RNAs find use in avariety of applications, including ribozyme, antisense, biophysical andbiochemical studies. Additionally, capped mRNA transcripts are used forapplications requiring protein synthesis such as in vivo expressionexperiments (using microinjection, transfection and infection), in vitrotranslation experiments and assays as well as various applications intherapeutics, diagnostics, vaccine development, labeling and detection.

Consequently, there is a need in the industry for compositions andmethods that allow for large scale synthesis of mRNAs that are (a) lesslaborious than conventional methods, (b) eliminate or reducebi-directional initiation during transcription, (c) result in higheryields of mRNA, at a (d) reduced cost compared to current methods, (e)reduces production of heterogeneous products with different 5′-sequencesand (f) does not require additional enzymatic reactions to incorporateCap 1 and Cap 2 structures into the synthesized mRNA. There is also aneed for the synthesis of various mRNAs containing modified and/orunnatural nucleosides, carrying specific modifications and/or affinitytags such as fluorescent dyes, a radioisotope, a mass tag and/or onepartner of a molecular binding pair such as biotin at or near the 5′ endof the molecule.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions for synthesizing 5′-cappedRNAs. In one aspect of the present invention, the initiating cappedoligonucleotide primers comprise the general structure of Formula I:

wherein

-   -   each of B₁ through B₁₀ is independently a natural, modified or        unnatural nucleoside base;    -   M is 0 or 1;    -   L is 0 or 1;    -   q₁ is 1;    -   each of q₂ through q₉ is independently 0 or 1;    -   R₁ is H or methyl;    -   R₂ and R₃ are independently H, OH, alkyl, O-alkyl, halogen,        amine, azide, a linker or a detectable marker;    -   each of X₁ through X₁₃ is independently O or S;    -   each of Y₁ through Y₁₃ is independently OH, SH, BH₃, aryl,        alkyl, O-alkyl or O-aryl;    -   each of Z₁ through Z₂₂ is independently O, S, NH, CH₂,        C(halogen)₂ or CH(halogen); and    -   each of R₄ through R₁₂ are independently H, OH, OMe, linker or a        detectable marker.

In another aspect of the present invention, there are provided RNAmolecules comprising a Cap containing initiating oligonucleotide primerof Formula I, pharmaceutical compositions comprising such RNA, cellscontaining such RNA and cells containing a protein or peptide translatedfrom such RNA.

In yet another aspect of the present invention, there are providedmethods for synthesizing RNA molecules comprising a Cap containinginitiating oligonucleotide primer of Formula I into a mixture comprisinga polynucleotide template and an RNA polymerase under conditionsconducive to transcription by the RNA polymerase of the polynucleotidetemplate, and thereafter incubating the resulting mixture for a timesufficient to allow for transcription of said template.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary method of preparing 7-methylguanosine5-diphosphate (pp^(7m)G) from guanosine 5′-diphosphate;

FIG. 2 shows an exemplary method of preparing 7-methylguanosine5′-diphosphate imidazolide (Im-pp^(7m)G) from pp^(7m)G;

FIG. 3 shows an exemplary method of preparing 3′-O-methylguanosine5′-phosphate (pG_(3′Ome)) from 3′-O-methylguanosine;

FIG. 4 shows an exemplary method of preparing 3′-O-methylguanosine5′-phosphorimidazolide (Im-pG_(3′Ome)) from pG_(3′Ome);

FIG. 5 shows an exemplary method of preparing of 3′-O-methylguanosine5′-diphosphate (ppG_(3′Ome)) from Im-pG_(3′Ome);

FIG. 6 shows an exemplary method for preparing7-methyl-3′-O-methylguanosine 5-diphosphate (pp^(7m)G_(3′Ome)) fromppG_(3′Ome);

FIG. 7 shows an exemplary method for preparing7-methyl-3′-O-methylguanosine 5-diphosphate imidazolide(Im-pp^(7m)G_(3′Ome)) from pp^(7m)G_(3′Ome);

FIG. 8 shows a general procedure for the preparation of pN_(2′-OR1)pNoligonucleotides (R₁═H or Me);

FIG. 9 shows a general procedure for the synthesis of initiatingoligonucleotides with Cap 0, Cap 1 or Cap 2 structures;

FIG. 10A shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is guanine;M is 0; L is 1; q₁ through q₉ are 0; R₁ is H; R₂ is H; R₃ is O-methyl;X₁ is O; X₂ is O; X₁₃ is O; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₂₂ isO;

FIG. 10B shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is guanine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is H; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH; Y₂ isOH; Y₄ is OH; Y₁₃ is OH; Z₀ is O, Z₁ is O; Z₂ is O; Z₄ is O; Z₅ is O;Z₂₂ is O; R₄ is O-methyl;

FIG. 10C shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is guanine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is O-methyl; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH;Y₂ is OH; Y₄ is OH; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ isO; Z₂₂ is O; R₄ is O-methyl;

FIG. 10D shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is adenine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is H; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH; Y₂ isOH; Y₄ is OH; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ is O;Z₂₂ is O; R₄ is O-methyl;

FIG. 10E shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is adenine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is O-methyl; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH;Y₂ is OH; Y₄ is OH; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ isO; Z₂₂ is O; R₄ is O-methyl;

FIG. 10F shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is cytosine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is H; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH; Y₂ isOH; Y₄ is OH; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ is O;Z₂₂ is O; R₄ is O-methyl;

FIG. 10G shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is cytosine;B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ through q₉ are 0; R₁ is H;R₂ is H; R₃ is O-methyl; X₁ is O; X₂ is O; X₄ is O; X₁₃ is O; Y₁ is OH;Y₂ is OH; Y₄ is OH; Y₁₃ is OH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ isO; Z₂₂ is O; R₄ is O-methyl;

FIG. 10H shows the structure of initiating capped oligonucleotideprimers used in examples according to Formula I wherein: B₁ is adenine;B₂ is guanine; B₁₀ is guanine; M is 0; L is 1; q₁ is 1; q₂ is 1; q₃through q₉ are 0; R₁ is H; R₂ is H; R₃ is O-methyl; X₁ is O; X₂ is O; X₄is O; X₅ is O; X₁₃ is O; Y₁ is OH; Y₂ is OH; Y₄ is OH; Y₅ is OH; Y₁₃ isOH; Z₀ is O; Z₁ is O; Z₂ is O; Z₄ is O; Z₅ is O; Z₆ is O; Z₂₂ is O; R₄is O-methyl; R₅ is O-methyl;

FIG. 11 shows the luciferase activity of mRNAs co-transcriptionallycapped mRNAs;

FIGS. 12A-12H collectively show the capping efficiency of mRNAsco-transcriptionally capped with 12A) ARCA, 12B) ^(m7)GpppG_(2′Ome)pG,12C) ^(m7)G_(3′Ome)pppG_(2′Ome)pG, 12D) ^(m7)GpppA_(2′Ome)pG, 12E)^(m7)G_(3′Ome)pppA_(2′-OMe)pG, 12F) ^(m7)GpppC_(2′Ome)pG, 12G)^(m7)G_(3′Ome)pppC_(2′Ome)pG, 12H)^(m7)G_(3′Ome)pppA_(2′Ome)pG_(2′Ome)pG. Capping efficiency in FIGS.11B-11G is equal to or significantly exceeds that observed in FIG. 11A;and

FIGS. 13A-13D collectively show the capping efficiency and fidelity ofinitiation of mRNAs co-transcriptionally capped with^(m7)GpppA_(2′Ome)pG on a transcription template, wherein FIG. 13Aillustrates the use of 2′-deoxythymidine and 2′-deoxycytidine residuesat template positions +1 and +2 using Primer/NTP formulation 2; FIG. 13Billustrates the use of 2′-deoxythymidine and 2′-deoxycytidine residuesat template positions +1 and +2 using Primer/NTP formulation 3; FIG. 13Cillustrates the use of 2′-deoxycytidine residues at template positions+1 and +2 using Primer/NTP formulation 2; and FIG. 13D illustrates theuse of 2′-deoxycytidine residues at template positions +1 and +2 usingPrimer/NTP formulation 3.

FIGS. 14A and 14B collectively show comparison of translation indifferentiated THP-1 cells of mRNA made with m7GpppA2′OmepG initiatingcapped oligonucleotide on a transcription template with2′-deoxythymidine and 2′-deoxycytidine residues at template positions +1and +2 vs. a transcription template with cytidine residues at templatepositions +1 and +2.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all terms used herein have the same meaning asare commonly understood by one of skill in the art to which thisinvention belongs. All patents, patent applications and publicationsreferred to throughout the disclosure herein are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail.

As used herein in connection with numerical values, the term“approximately” or “about” means plus or minus 30% of the indicatedvalue, including all values within the defined range, including thestated value.

As used herein, the terms “nucleic acid,” “nucleotide sequence,” or“nucleic acid sequence” refer to an oligonucleotide, polynucleotide, orany fragment thereof, any ribo or deoxyribo derivatives and to naturallyoccurring or synthetic molecules containing natural and/or modifiednucleotide residues and internucleotide linkages. These phrases alsorefer to DNA or RNA of natural (e.g., genomic) or synthetic origin whichmay be single-stranded, double-stranded, triple-stranded ortetra-stranded and may represent the sense or the antisense strand, orto any DNA-like or RNA-like material. An “RNA equivalent,” in referenceto a DNA sequence, is composed of the same linear sequence ofnucleotides as the reference DNA sequence with the exception that all ormost occurrences of the nitrogenous base thymine are replaced withuracil, and the sugar backbone is composed of ribose instead of2′-deoxyribose. Additional alternative nucleic acid backbones suitablefor the methods and compositions provided herein include but are notlimited to phosphorothioate, phosphoroselenoate, alkyl phosphotriester,aryl phosphotriester, alkyl phosphonate, aryl phosphonate,phosphoboronate, morpholino nucleic acid (MNA), locked nucleic acids(LNA), peptide nucleic acids (PNA).

As used herein, the term “primer” or “oligonucleotide primer” refers toa ribo- or deoxyribo- or chimeric ribo/deoxyribo-oligonucleotide, singlestranded, may be naturally occurring or synthetic, and usually include asequence of between about 2 to about 10 nucleotides, about 3 to about 8nucleotides or about 3 to about 5 nucleotides. Oligonucleotide primersmay contain one or more modification groups. Oligonucleotide primers mayinclude RNA, DNA, and/or other modified nucleosides. The skilled artisanis capable of designing and preparing oligonucleotide primers that areappropriate for transcription of DNA template sequence.

As used herein, the terms “initiating capped oligonucleotide analogs” or“initiating capped oligonucleotide primers” refer to an initiatingoligonucleotide primer containing Cap 0, Cap 1, Cap 2 or TMG-Capstructure on 5′-end of the primer. The capped primer has an unmodifiedor open 3′-OH group and it may be extended by RNA polymerase through theincorporation of an NTP onto the 3′-end of the primer. It is able toinitiate in vitro transcription under the control of a promoter in atranscription system containing necessary components: DNA template (e.g.DNA plasmid), RNA polymerase, nucleoside 5′-triphosphates andappropriate buffer. Also used herein, “initiating primer” or “initiatingoligonucleotide primer” refers to an oligonucleotide, carrying aterminal 3′-OH group that is a valid substrate for RNA polymerase. Incertain embodiments, the initiating oligonucleotide primer is asubstrate for RNA polymerase and may be elongated by incorporation ofNTP onto the 3′-end of the primer. The initiating oligonucleotide primeris complementary to the DNA template at the initiation site.

As used herein, the term “unsubstituted” or “unmodified” in the contextof the initiating capped oligonucleotide primer and NTPs refers to aninitiating capped oligonucleotide primer and NTPs that have not beenmodified.

As used herein, the term “modified initiating capped oligonucleotideprimer” refers to an initiating capped oligonucleotide primer thatcontains one or more additional modification groups.

As used herein, the term “modification group” refers to any chemicalmoiety that may be attached to the initiating primer at locations, whichinclude, but are not limited to, the sugar, nucleoside base,triphosphate bridge, and/or internucleotide phosphate (e.g., U.S. PatentApplication No. 20070281308). The modification group of an initiatingcapped oligonucleotide primer may be a group of any nature compatiblewith the process of transcription.

As used herein, the term “internucleotide linkage” refers to the bond orbonds that connect two nucleosides of an oligonucleotide primer ornucleic acid and may be a natural phosphodiester linkage or modifiedlinkage.

As used herein, the term “label” or “detectable label” refers to anycompound or combination of compounds that may be attached or otherwiseassociated with a molecule so that the molecule can be detected directlyor indirectly by detecting the label. A detectable label can be aradioisotope (e.g., carbon, phosphorus, iodine, indium, sulfur, tritiumetc.), a mass isotope (e.g., H², C¹³ or N¹⁵), a dye or fluorophore(e.g., cyanine, fluorescein or coumarin), a hapten (e.g., biotin) or anyother agent that can be detected directly or indirectly.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where initiating capped oligonucleotide primer anneals to aDNA template under appropriately stringent conditions during atranscription reaction. Hybridizations to DNA are conducted with aninitiating capped oligonucleotide primer which, in certain embodiments,is 3-10 nucleotides in length including the 5′-5′ inverted capstructure. Nucleic acid hybridization techniques are well known in theart (e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press, Plainview, N.Y. (1989);Ausubel, F. M., et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Secaucus, N.J. (1994)).

As used herein, the term “complement,” “complementary,” or“complementarity” in the context of a complex of an initiating cappedoligonucleotide primer and a DNA template refers to standardWatson/Crick base pairing rules. For example, the sequence“5′-A-G-T-C-3′” is complementary to the sequence “3′-T-C-A-G-5′.”Certain non-natural or synthetic nucleotides may be included in thenucleic acids described herein; these include but not limited to, baseand sugar modified nucleosides, nucleotides, and nucleic acids, such asinosine, 7-deazaguanosine, 2′-O-methylguanosine,2′-fluoro-2′-deoxycytidine, pseudouridine, Locked Nucleic Acids (LNA),and Peptide Nucleic Acids (PNA). Complementarity does not need to beperfect; duplexes may contain mismatched base pairs, degenerative, orunmatched nucleotides. Those skilled in the art can determine duplexstability empirically considering a number of variables including, forexample, the length of the oligonucleotide, base composition andsequence of the oligonucleotide, incidence of mismatched base pairs,ionic strength, components of the hybridization buffer and reactionconditions.

Complementarity may be “complete” or “total” where all of the nucleotidebases of two nucleic acid strands are matched according to recognizedbase pairing rules, it may be “partial” in which only some of thenucleotide bases of an initiating capped oligonucleotide primer and aDNA target are matched according to recognized base pairing rules or itmay be “absent” where none of the nucleotide bases of two nucleic acidstrands are matched according to recognized base pairing rules. Thedegree of complementarity between of an initiating cappedoligonucleotide primer and a DNA template may have a significant effecton the strength of hybridization between the initiating cappedoligonucleotide and the DNA template and correspondingly the efficiencyof the reaction. The term complementarity may also be used in referenceto individual nucleotides. For example, a particular nucleotide withinan oligonucleotide may be noted for its complementarity, or lackthereof, to a nucleotide within another strand, in contrast orcomparison to the complementarity between the rest of an initiatingcapped oligonucleotide primer and DNA strand.

As used herein the term “complete”, “total” or “perfectly” complementarymeans that each of the nucleotide bases of an initiating cappedoligonucleotide primer and a DNA target are matched exactly according torecognized base pairing rules.

As used herein, the term “substantially complementary” refers to twosequences that hybridize under stringent hybridization conditions. Thoseskilled in the art will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences may comprise a contiguous sequenceof bases that do not hybridize to a target sequence and may bepositioned 3′ or 5′ to a contiguous sequence of bases that hybridizeunder stringent hybridization conditions to the target sequence.

As used herein, the term “specific” when used in reference to aninitiating capped oligonucleotide primer sequence and its ability tohybridize to a DNA template is a sequence that has at least 50% sequenceidentity with a portion of the DNA template when the initiating cappedoligonucleotide primer and DNA strand are aligned. Higher levels ofsequence identity that may be preferred include at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, and mostpreferable 100% sequence identity.

As used herein, the term “nucleoside” includes all naturally occurringnucleosides, including all forms of nucleoside bases and furanosidesfound in nature. Base rings most commonly found in naturally occurringnucleosides are purine and pyrimidine rings. Naturally occurring purinerings include, for example, adenine, guanine, and N⁶-methyladenine.Naturally occurring pyrimidine rings include, for example, cytosine,thymine, 5-methylcytosine, pseudouracyl. Naturally occurring nucleosidesfor example include, but are not limited to, ribo, 2′-O-methyl or2′-deoxyribo derivatives of adenosine, guanosine, cytidine, thymidine,uridine, inosine, 7-methylguanosine or pseudouridine.

As used herein, the terms “nucleoside analogs,” “modified nucleosides,”or “nucleoside derivatives” include synthetic nucleosides as describedherein. Nucleoside derivatives also include nucleosides having modifiedbase or/and sugar moieties, with or without protecting groups andinclude, for example, 2′-deoxy-2′-fluorouridine, 5-fluorouridine and thelike. The compounds and methods provided herein include such base ringsand synthetic analogs thereof, as well as unnaturalheterocycle-substituted base sugars, and acyclic substituted basesugars. Other nucleoside derivatives that may be utilized with thepresent invention include, for example, LNA nucleosides,halogen-substituted purines (e.g., 6-fluoropurine), halogen-substitutedpyrimidines, N⁶-ethyladenine, N⁴-(alkyl)-cytosines, 5-ethylcytosine, andthe like (U.S. Pat. No. 6,762,298).

As used herein, the terms “universal base,” “degenerate base,”“universal base analog” and “degenerate base analog” include, forexample, a nucleoside analog with an artificial base which is, incertain embodiments, recognizable by RNA polymerase as a substitute forone of the natural NTPs (e.g., ATP, UTP, CTP and GTP) or other specificNTP. Universal bases or degenerate bases are disclosed in Loakes, D.,Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C., et. al.,Nucleic Acids Res., 33:1532-1543 (2005); Kincaid, K., et. al., NucleicAcids Res., 33:2620-2628 (2005); Preparata, F P, Oliver, J S, J. Comput.Biol. 753-765 (2004); and Hill, F., et. al., Proc Natl Acad. Sci. USA,95:4258-4263 (1998)).

As used herein, the term “modified NTP” refers to a nucleoside5′-triphosphate having a chemical moiety group bound at any position,including the sugar, base, triphosphate chain, or any combination ofthese three locations. Examples of such NTPs can be found, for examplein “Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnologyand Biological Applications,” Vaghefi, M., ed., Taylor and Francis, BocaRaton (2005).

As used herein, the term “modified oligonucleotide” includes, forexample, an oligonucleotide containing a modified nucleoside, a modifiedinternucleotide linkage, or having any combination of modifiednucleosides and internucleotide linkages. Examples of oligonucleotideinternucleotide linkage modifications including phosphorothioate,phosphotriester and methylphosphonate derivatives (Stec, W. J., et al.,Chem. Int. Ed. Engl., 33:709-722 (1994); Lebedev, A. V., et al., E.,Perspect. Drug Discov. Des., 4:17-40 (1996); and Zon, et al., U.S.Patent Application No. 20070281308). Other examples of internucleotidelinkage modifications may be found in Waldner, et al., Bioorg. Med.Chem. Letters 6:2363-2366 (1996).

The term “promoter” as used herein refers to a region of dsDNA templatethat directs and controls the initiation of transcription of aparticular DNA sequence (e.g. gene). Promoters are located on the samestrand and upstream on the DNA (towards the 5′ region of the sensestrand). Promoters are typically immediately adjacent to (or partiallyoverlap with) the DNA sequence to be transcribed. Nucleotide positionsin the promoter are designated relative to the transcriptional startsite, where transcription of DNA begins (position +1). The initiatingoligonucleotide primer is complementary to initiation site of promotersequence (which, in certain embodiments, is at positions +1 and +2 and,in the case of initiating tetramers, at positions +1, +2 and +3).

As used herein, the terms “transcription” or “transcription reaction”refers to methods known in the art for enzymatically making RNA that iscomplementary to DNA template, thereby producing the number of RNAcopies of a DNA sequence. The RNA molecule synthesized in transcriptionreaction called “RNA transcript”, “primary transcript” or “transcript”.Transcription reaction involving the compositions and methods providedherein employs “initiating capped oligonucleotide primers”.Transcription of DNA template may be exponential, nonlinear or linear. ADNA template may be a double stranded linear DNA, a partially doublestranded linear DNA, circular double stranded DNA, DNA plasmid, PCRamplicon, a modified nucleic acid template which is compatible with RNApolymerase.

As used herein, the term “acyl” denotes the group —C(O)R^(a), whereR^(a) is hydrogen, lower alkyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, and the like.

As used herein, the term “substituted acyl” denotes the group—C(O)R^(a′), where R^(a′) is substituted lower alkyl, substitutedcycloalkyl, substituted heterocyclyl, substituted aryl, substitutedheteroaryl, and the like.

As used herein, the term “acyloxy” denotes the group —OC(O)R^(b), whereR^(b) is hydrogen, lower alkyl, substituted lower alkyl, cycloalkyl,substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, and the like.

As used herein, the term “alkyl” refers to a single bond chain ofhydrocarbons ranging, in some embodiments, from 1-20 carbon atoms, andranging in some embodiments, from 1-8 carbon atoms; examples includemethyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.

As used herein, the term “lower alkyl” refers to a straight chain or abranched chain of hydrocarbons ranging, in some embodiments, from 1-6carbon atoms, and ranging in some embodiments from 2-5 carbon atoms.Examples include ethyl, propyl, isopropyl, and the like.

As used herein, the term “alkenyl” refers to a straight-chain orbranched-chain hydrocarbyl, which has one or more double bonds and,unless otherwise specified, contains from about 2 to about 20 carbonatoms, and ranging in some embodiments from about 2 to about 10 carbonatoms, and ranging in some embodiments from about 2 to about 8 carbonatoms, and ranging in some embodiments from about 2 to about 6 carbonatoms. Examples of alkenyl radicals include vinyl, allyl,1,4-butadienyl, isopropenyl, and the like.

As used herein, the term “alkenylaryl” refers to alkenyl-substitutedaryl groups and “substituted alkenylaryl” refers to alkenylaryl groupsfurther bearing one or more substituents as set forth herein.

As used herein, the term “alkenylene” refers to divalent straight-chainor branched-chain hydrocarbyl groups having at least one carbon-carbondouble bond, and typically containing 2-20 carbon atoms, and ranging insome embodiments from 2-12 carbon atoms, and ranging in some embodimentsfrom 2-8 carbon atoms, and “substituted alkenylene” refers to alkenylenegroups further bearing one or more substituents as set forth herein.

As used herein, the term “alkylene” refers to divalent hydrocarbyl groupcontaining 1-20 carbon atoms, and ranging in some embodiments from 1-15carbon atoms, straight-chain or branched-chain, from which two hydrogenatoms are taken from the same carbon atom or from different carbonatoms. Examples of alkylene include, but are not limited to, methylene(—CH₂—), ethylene (—CH₂CH₂—), and the like.

As used herein, the term “alkynyl” refers to a straight-chain orbranched-chain hydrocarbyl, which has one or more triple bonds andcontains from about 2-20 carbon atoms, and ranging in some embodimentsfrom about 2-10 carbon atoms, and ranging in some embodiments from about2-8 carbon atoms, and ranging in some embodiments from about 2-6 carbonatoms. Examples of alkynyl radicals include ethynyl, propynyl(propargyl), butynyl, and the like.

As used herein, the term “alkynylaryl” refers to alkynyl-substitutedaryl groups and “substituted alkynylaryl” refers to alkynylaryl groupsfurther bearing one or more substituents as set forth herein.

As used herein, the term “alkoxy” denotes the group —OR^(c), where R^(c)is lower alkyl, substituted lower alkyl, aryl, substituted aryl,aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, or substitutedcycloheteroalkyl as defined.

As used herein, the term “lower alkoxy” denotes the group —OR^(d), whereR^(d) is lower alkyl.

As used herein, the term “alkylaryl” refers to alkyl-substituted arylgroups and “substituted alkylaryl” refers to alkylaryl groups furtherbearing one or more substituents as set forth herein.

As used herein, the term “alkylthio” refers to the group —S—R^(h), whereR^(h) is alkyl.

As used herein, the term “substituted alkylthio” refers to the group—S—R^(i), where R^(i) is substituted alkyl.

As used herein, the term “alkynylene” refers to divalent straight-chainor branched-chain hydrocarbyl groups having at least one carbon-carbontriple bond, and typically having in the range of about 2-12 carbonatoms, and ranging in some embodiments from about 2-8 carbon atoms, and“substituted alkynylene” refers to alkynylene groups further bearing oneor more substituents as set forth herein.

As used herein, the term “amido” denotes the group —C(O)NR^(j)R^(j′),where R^(j) and R^(j′) may independently be hydrogen, lower alkyl,substituted lower alkyl, alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, or substituted heteroaryl.

As used herein, the term “substituted amido” denotes the group—C(O)NR^(k)R^(k′), where R^(k) and R^(k′) are independently hydrogen,lower alkyl, substituted lower alkyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl, provided, however, that at leastone of R^(k) and R^(k′) is not hydrogen. R^(k)R^(k′) in combination withthe nitrogen may form an optionally substituted heterocyclic orheteroaryl ring.

As used herein, the term “amino” or “amine” denotes the group—NR^(n)R^(n′), where R^(n) and R^(n′) may independently be hydrogen,lower alkyl, substituted lower alkyl, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, or substituted heteroaryl as definedherein. A “divalent amine” denotes the group —NH—. A “substituteddivalent amine” denotes the group —NR— where R is lower alkyl,substituted lower alkyl, alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, or substituted heteroaryl.

As used herein, the term “substituted amino” or “substituted amine”denotes the group —NR^(p)R^(p′), where R^(p) and R^(p′) areindependently hydrogen, lower alkyl, substituted lower alkyl, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, provided, however, that at least one of R^(p) and R^(p′) isnot hydrogen. R^(p)R^(p′) in combination with the nitrogen may form anoptionally substituted heterocyclic, or heteroaryl ring.

As used herein, the term “aroyl” refers to aryl-carbonyl species such asbenzoyl and “substituted aroyl” refers to aroyl groups further bearingone or more substituents as set forth herein.

As used herein, the term “aryl” alone or in combination refers tophenyl, naphthyl or fused aromatic heterocyclic optionally with acycloalkyl of 5-10 ring members, and in some embodiments 5-6, ringmembers and/or optionally substituted with 1 to 3 groups or substituentsof halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,acyloxy, aryloxy, heteroaryloxy, amino optionally mono- ordi-substituted with alkyl, aryl or heteroaryl groups, amidino, ureaoptionally substituted with alkyl, aryl, heteroaryl or heterocyclylgroups, aminosulfonyl optionally N-mono- or N,N-di-substituted withalkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino,heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, or the like.

As used herein, the term “aryloxy” denotes the group —OAr, where Ar isan aryl, or substituted aryl group.

As used herein, the term “carbocycle” refers to a saturated,unsaturated, or aromatic group having a single ring or multiplecondensed rings composed of linked carbon atoms. The ring(s) canoptionally be unsubstituted or substituted with, e.g., halogen, loweralkyl, alkoxy, alkylthio, acetylene (—C≡CH), amino, amido, azido,carboxyl, hydroxyl, aryl, aryloxy, heterocycle, heteroaryl, substitutedheteroaryl, nitro (—NO₂), cyano (—CN), thiol (—SH), sulfamido(—S(O)₂NH₂, and the like.

As used herein, the term “guanidinyl” denotes the group —N═C(NH₂)₂ and“substituted guanidinyl” denotes the group —N═C(NR₂)₂, where each R isindependently H, alkyl, substituted alkyl, aryl, or substituted aryl asset forth herein.

As used herein, the term “halo” or “halogen” refers to all halogens,i.e., chloro (Cl), fluoro (F), bromo (Br), and iodo (I).

As used herein, the term “heteroaryl” refers to a monocyclic aromaticring structure containing 5 or 6 ring atoms, or a bicyclic aromaticgroup having 8-10 atoms, containing one or more, and in some embodiments1-4, and in some embodiments 1-3, and in some embodiments 1-2heteroatoms independently selected from the group O, S, and N, andoptionally substituted with 1-3 groups or substituents of halo, hydroxy,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy,heteroaryloxy, amino optionally mono- or di-substituted with alkyl, arylor heteroaryl groups, amidino, urea optionally substituted with alkyl,aryl, heteroaryl, or heterocyclyl groups, aminosulfonyl optionallyN-mono- or N,N-di-substituted with alkyl, aryl or heteroaryl groups,alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino,alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or thelike. Heteroaryl is also intended to include oxidized S or N, such assulfinyl, sulfonyl, and N-oxide of a tertiary ring nitrogen. A carbon ornitrogen atom is the point of attachment of the heteroaryl ringstructure such that a stable aromatic ring is retained. Examples ofheteroaryl groups are phthalimide, pyridinyl, pyridazinyl, pyrazinyl,quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl,oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl,tetrazolyl, imidazolyl, triazinyl, furanyl, benzofuryl, indolyl, and thelike. A substituted heteroaryl contains a substituent attached at anavailable carbon or nitrogen to produce a stable compound.

As used herein, the term “substituted heteroaryl” refers to aheterocycle optionally mono or poly substituted with one or morefunctional groups, e.g., halogen, lower alkyl, lower alkoxy, alkylthio,acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle,substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano,thiol, sulfamido, and the like.

As used herein, the term “heterocycle” refers to a saturated,unsaturated, or aromatic group having a single ring (e.g., morpholino,pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyl,quinoxalyl, quinolinyl, indolizinyl or benzo[b]thienyl) and havingcarbon atoms and at least one hetero atom, such as N, O or S, within thering, which can optionally be unsubstituted or substituted with, e.g.,halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido,carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substitutedhetaryl, nitro, cyano, thiol, sulfamido, and the like.

As used herein, the term “substituted heterocycle” refers to aheterocycle substituted with 1 or more, e.g., 1, 2, or 3, substituentsselected from the group consisting of optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, halo,hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryl,substituted aryl, aryloxy, heteroaryloxy, amino, amido, amidino, ureaoptionally substituted with alkyl, aryl, heteroaryl or heterocyclylgroups, aminosulfonyl optionally N-mono- or N,N-di-substituted withalkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino,heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, acyl, carboxyl, heterocycle, substitutedheterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfonamido, and oxo, attached at any available point to produce astable compound.

As used herein, the term “hydrocarbyl” refers to any organic radicalwhere the backbone thereof comprises carbon and hydrogen only. Thus,hydrocarbyl embraces alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, alkylaryl, arylalkyl, arylalkenyl, alkenylaryl, arylalkynyl,alkynylaryl, and the like.

As used herein, the term “substituted hydrocarbyl” refers to any of theabove-referenced hydrocarbyl groups further bearing one or moresubstituents selected from hydroxy, hydrocarbyloxy, substitutedhydrocarbyloxy, alkylthio, substituted alkylthio, arylthio, substitutedarylthio, amino, alkylamino, substituted alkylamino, carboxy, —C(S)SR,—C(O)SR, —C(S)NR₂, where each R is independently hydrogen, alkyl orsubstituted alkyl, nitro, cyano, halo, —SO₃M or —OSO₃M, where M is H,Na, K, Zn, Ca, or meglumine, guanidinyl, substituted guanidinyl,hydrocarbyl, substituted hydrocarbyl, hydrocarbylcarbonyl, substitutedhydrocarbylcarbonyl, hydrocarbyloxycarbonyl, substitutedhydrocarbyloxycarbonyl, hydrocarbylcarbonyloxy, substitutedhydrocarbylcarbonyloxy, acyl, acyloxy, heterocyclic, substitutedheterocyclic, heteroaryl, substituted heteroaryl, heteroarylcarbonyl,substituted heteroarylcarbonyl, carbamoyl, monoalkylcarbamoyl,dialkylcarbamoyl, arylcarbamoyl, a carbamate group, a dithiocarbamategroup, aroyl, substituted aroyl, organosulfonyl, substitutedorganosulfonyl, organosulfinyl, substituted alkylsulfinyl,alkylsulfonylamino, substituted alkylsulfonylamino, arylsulfonylamino,substituted arylsulfonylamino, a sulfonamide group, sulfuryl, and thelike, including two or more of the above-described groups attached tothe hydrocarbyl moiety by such linker/spacer moieties as —O—, —S—, —NR—,where R is hydrogen, alkyl or substituted alkyl, —C(O)—, —C(S)—,—C(═NR′)—, —C(═CR′₂)—, where R′ is alkyl or substituted alkyl, —O—C(O)—,—O—C(O)—O—, —O—C(O)—NR— (or —NR—C(O)—O—), —NR—C(O)—, —NR—C(O)—NR—,—S—C(O)—, —S—C(O)—O—, —S—C(O)—NR—, —O—S(O)₂—, —O—S(O)₂—O—, —O—S(O)₂—NR—,—O—S(O)—, —O—S(O)—O—, —O—S(O)—NR—, —O—NR—C(O)—, —O—NR—C(O)—O—,—O—NR—C(O)—NR—, —NR—O—C(O)—, —NR—O—C(O)—O—, —NR—O—C(O)—NR—, —O—NR—C(S)—,—O—NR—C(S)—O—, —O—NR—C(S)—NR—, —NR—O—C(S)—, —NR—O—C(S)—O—,—NR—O—C(S)—NR—, —O—C(S)—, —O—C(S)—O—, —O—C(S)—NR— (or —NR—C(S)—O—),—NR—C(S)—, —NR—C(S)—NR—, —S—S(O)₂—, —S—S(O)₂—O—, —S—S(O)₂—NR—,—NR—O—S(O)—, —NR—O—S(O)—O—, —NR—O—S(O)—NR—, —NR—O—S(O)₂—,—NR—O—S(O)₂—O—, —NR—O—S(O)₂—NR—, —O—NR—S(O)—, —O—NR—S(O)—O—,—O—NR—S(O)—NR—, —O—NR—S(O)₂—O—, —O—NR—S(O)₂—NR—, —O—NR—S(O)₂—,—O—P(O)R₂—, —S—P(O)R₂—, or —NR—P(O)R₂—, where each R is independentlyhydrogen, alkyl or substituted alkyl, and the like.

As used herein, the term “hydroxyl” or “hydroxy” refers to the group—OH.

As used herein, the term “oxo” refers to an oxygen substituent doublebonded to the attached carbon.

As used herein, the term “sulfinyl” denotes the group —S(O)—.

As used herein, the term “substituted sulfinyl” denotes the group—S(O)R^(t), where R^(t) is lower alkyl, substituted lower alkyl,cycloalkyl, substituted cycloalkyl, cycloalkylalkyl, substitutedcycloalkylalkyl, heterocyclyl, substituted heterocyclyl,heterocyclylalkyl, substituted hetereocyclylalkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heteroaralkyl, substitutedheteroaralkyl, aralkyl, or substituted aralkyl.

As used herein, the term “sulfonyl” denotes the group —S(O)₂—.

As used herein, the term “substituted sulfonyl” denotes the group—S(O)₂R^(t), where R^(t) is lower alkyl, substituted lower alkyl,cycloalkyl, substituted cycloalkyl, cycloalkylalkyl, substitutedcycloalkylalkyl, heterocyclyl, substituted heterocyclyl,heterocyclylalkyl, substituted hetereocyclylalkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heteroaralkyl, substitutedheteroaralkyl, aralkyl, or substituted aralkyl.

As used herein, the term “sulfuryl” denotes the group —S(O)₂—.

The present invention provides methods and compositions for synthesizing5′Capped RNAs wherein the initiating capped oligonucleotide primers havethe general form ^(m7)Gppp[N_(2′Ome)]_(n[)N]_(m) wherein ^(m7)G isN7-methylated guanosine or any guanosine analog, N is any natural,modified or unnatural nucleoside “n” can be any integer from 1 to 4 and“m” can be an integer from 1 to 9. In one aspect of the invention, theinitiating capped oligonucleotide primers have the structure of FormulaI:

wherein:

-   -   each of B₁ through B₁₀ is independently a natural, modified or        unnatural nucleoside base;    -   M is 0 or 1;    -   L is 0 or 1;    -   q1 is 1;    -   each of q₂ through q₉ is independently 0 or 1;    -   R₁ is H or methyl;    -   R₂ and R₃ are independently H, OH, alkyl, O-alkyl, amine, azide,        halogen, a linker or a detectable marker;    -   each of X₁ through X₁₃ is independently O or S;    -   each of Y₁ through Y₁₃ is independently OH, SH, BH₃, aryl,        alkyl, O-alkyl or O-aryl;    -   each of Z₁ through Z₂₂ is independently O, S, NH, CH₂,        C(halogen)₂ or CH(halogen) and    -   each of R₄ through R₁₂ are independently, H, OH, OMe or a        detectable marker.

In certain embodiments the initiating capped oligonucleotide primer is atrimer (q²-q⁹=0), tetramer (q³-q⁹=0), pentamer (q⁴-q⁹=0), hexamer(q⁵-q⁹=0), heptamer (q⁶-q⁹=0), octamer (q⁷-q⁹=0), nanomer (q⁸-q⁹=0),decamer (q⁹=0) or an undecamer. A number of examples of the initiatingcapped oligonucleotide trimer primers are presented in Table I below:

TABLE I Sequence of Initiating Capped Oligonucleotide Primer m⁷GpppApAm⁷GpppApC m⁷GpppApG m⁷GpppApU m⁷GpppCpA m⁷GpppCpC m⁷GpppCpG m⁷GpppCpUm⁷GpppGpA m⁷GpppGpC m⁷GpppGpG m⁷GpppGpU m⁷GpppUpA m⁷GpppUpC m⁷GpppUpGm⁷GpppUpU m⁷G_(3′Ome)pppApA m⁷G_(3′Ome)pppApC m⁷G_(3′Ome)pppApGm⁷G_(3′Ome)pppApU m⁷G_(3′Ome)pppCpA m⁷G_(3′Ome)pppCpC m⁷G_(3′Ome)pppCpGm⁷G_(3′Ome)pppCpU m⁷G_(3′Ome)pppGpA m⁷G_(3′Ome)pppGpC m⁷G_(3′Ome)pppGpGm⁷G_(3′Ome)pppGpU m⁷G_(3′Ome)pppUpA m⁷G_(3′Ome)pppUpC m⁷G_(3′Ome)pppUpGm⁷G_(3′Ome)pppUpU m⁷G_(3′Ome)pppA_(2′Ome)pA m⁷G_(3′Ome)pppA_(2′Ome)pCm⁷G_(3′Ome)pppA_(2′Ome)pG m⁷G_(3′Ome)pppA_(2′Ome)pUm⁷G_(3′Ome)pppU_(2′Ome)pA m⁷G_(3′Ome)pppU_(2′Ome)pCm⁷G_(3′Ome)pppU_(2′Ome)pG m⁷G_(3′Ome)pppU_(2′Ome)pUm⁷G_(3′Ome)pppG_(2′Ome)pA m⁷G_(3′Ome)pppG_(2′Ome)pCm⁷G_(3′Ome)pppG_(2′Ome)pG m⁷G_(3′Ome)pppG_(2′Ome)pUm⁷G_(3′Ome)pppU_(2′Ome)pA m⁷G_(3′Ome)pppU_(2′Ome)pCm⁷G_(3′Ome)pppU_(2′Ome)pG m⁷G_(3′Ome)pppU_(2′Ome)pU m⁷GpppA_(2′Ome)pAm⁷GpppA_(2′Ome)pC m⁷GpppA_(2′Ome)pG m⁷GpppA_(2′Ome)pU m⁷GpppC_(2′Ome)pAm⁷GpppC_(2′Ome)pC m⁷GpppC_(2′Ome)pG m⁷GpppC_(2′Ome)pU m⁷GpppG_(2′Ome)pAm⁷GpppG_(2′Ome)pC m⁷GpppG_(2′Ome)pG m⁷GpppG_(2′Ome)pU m⁷GpppU_(2′Ome)pAm⁷GpppU_(2′Ome)pC m⁷GpppU_(2′Ome)pG m⁷GpppU_(2′Ome)pU

Other initiating capped oligonucleotide primers encompassed by thisinvention include those primers having known or novel base analogues.Methods for synthesizing—initiating capped oligonucleotide primers areexemplified in the examples below.

Transcription

In Eukaryotes, transcription of messenger RNAs (mRNAs) is done by RNApolymerase II. This is a complicated multi-subunit enzyme with complexregulation. To carry out large scale transcription in vitro, researchescommonly use single subunit phage polymerases derived from T7, T3, SP6,K1-5, K1E, K1F or K11 bacteriophages. This family of polymerases hassimple, minimal promoter sequences of ˜17 nucleotides which require noaccessory proteins and have minimal constraints of the initiatingnucleotide sequence. While this application focuses on T7 RNA Polymerase(T7 RNAP), one skilled in the art would understand that this inventioncould be practiced with other RNA polymerases.

T7 RNAP exists in at least two protein states. The first is referred toas the “abortive complex” and is associated with transcriptionalinitiation. The second is a very processive conformation called the“elongation complex”. In vitro transcription can be broken into sixsteps: 1) binding of the RNA polymerase to the promoter sequence, 2)initiation of transcription, 3) non-processive elongation termedabortive transcription during which the polymerase frequently releasesthe DNA template and short abortive transcripts 4) conversion of theopen complex to the closed complex, 5) processive elongation and 6)transcriptional termination. A significant amount of RNA produced duringtranscription consists of short abortive fragments of ˜2-8 nucleotidesin length (Biochemistry 19:3245-3253 (1980); Nucleic Acids Res. 9:31-45(1981); Nucleic Acids Res. 15:8783-8798 (1987); Biochemistry27:3966-3974 (1988)). After synthesis of about 10-14 bases, RNApolymerases escape from abortive cycling, at the same time losingsequence-specific contacts with the promoter DNA, and forming aprocessive elongation complex, in which the RNA chain is extended in asequence-independent manner (J. Mol. Biol. 183:165-177(1985); Proc.Natl. Acad. Sci. U.S.A. 83:3614-3618(1986); Mol. Cell Biol. 7:3371-3379(1987)).

The consensus sequence for the most active Class III T7 promotersencompasses 17 bp of sequence upstream, and 6 bp downstream, of thetranscription start site (Cell 16:815-25. (1979)). The position of thefirst transcribed nucleotide is commonly referred to as the +1transcript nucleotide of the RNA, the second transcribed nucleotide as+2 transcript nucleotide and so on (Table 2). During transcription, thetwo strands are melted to form a transcription bubble and the bottomstrand of the duplex (shown 3′ to 5′ in Table 2) is the template fortranscription. For transcript nucleotides +3 and beyond, the templatestrand defines the identity of the transcribed nucleotides primarilythrough Watson-Crick base pairing interactions. Here the nucleotideencoding the first RNA transcript nucleotide is defined as the +1nucleotide of the template. In the example shown in Table 2, the +1transcript nucleotide is G and the +1 template nucleotide is C. Likewisethe +4 transcript nucleotide is A and the +4 template nucleotide is T.

Table 2

Position in transcript                  +1+2+3+4+5+6                    |||||| Transcript sequence                 pppGGGAGA                     

Promoter top strand 5′-TAATACGACTCACTATAGGGAGA . . . -3′ SEQ ID NO. 1Promoter bottom strand 3′-ATTATGCTGAGTGATATCCCTCT . . . -5′ SEQ ID No. 2                    |||||| Position in template                 +1+2+3+4+5+6

Unlike DNA polymerases, T7 RNAP initiates RNA synthesis in the absenceof a primer. The first step in initiation is called de novo RNAsynthesis, in which RNA polymerase recognizes a specific sequence on theDNA template, selects the first pair of nucleotide triphosphatescomplementary to template residues at positions +1 and +2, and catalyzesthe formation of a phosphodiester bond to form a dinucleotide. Theinitiating nucleotides have lower affinities for the polymerase thanthose used during elongation. The Kd value is 2 mM for the firstinitiating NTP and 80 μM for the second, whereas the Kd is approximately5 μM for NTPs during elongation (J. Mol. Biol. (2007) 370, 256-268). Ithas been found that de novo synthesis is the rate-limiting step duringtranscription. T7 RNAP exhibits a strong bias for GTP as the initiatingnucleotide (J. Biol. Chem. 248: 2235-2244 (1973)). Among the 17 T7promoters in the genome, 15 initiate with GTP (and 13 with pppGpG),whereas there is no obvious NTP preference during transcriptionelongation (J. Mol. Biol. 370:256-268 (2007)). T7 RNA polymeraseinitiates poorly on promoters encoding A at position +1; transcriptioninstead initiates predominantly with an encoded G at position +2 (J.Biol. Chem. 278:2819-2823 (2003)).

During de novo RNA synthesis, binding of the initiating nucleotides isachieved primarily by the free energy created from base stacking,specific interactions between the polymerase residues, the guaninemoieties of the initiating nucleotides and base complementarityinteractions (J. Mol. Biol. 370:256-268 (2007)).

It is known that T7 RNAP can also initiate with short oligonucleotideprimers. For example, it is known that 13 promoters in the T7 genomeinitiate with pppGpG (J. Mol. Biol. 370:256-268 (2007)). Several groupsshowed that T7 RNAP can initiate from dinucleotide primers (Biochemistry24:5716-5723 (1985)). Axelrod et al. showed that an uncapped GpAdinucleotide could initiate from +1 and +2 template nucleotides thatwere 2′-deoxycytidine and 2′-deoxythymidine, respectively (“CT”template) Their reaction conditions were 200 micromolar (μM) dimer and100 μM ATP, CTP, GTP and UTP. Their reaction mixture also contained 100μM 3′ dATP, 3′ dCTP 3′ dUTP or 50 μM 3′ dGTP. They observe only GpAinitiated RNAs and not a mixture of GpA initiated RNAs and 5′triphosphate RNAs from GTP initiation. This is likely due to thereaction conditions employed. 100 μM GTP is well below the 2 mM Kd of T7polymerase for the first initiating guanosine (J. Mol. Biol. (2007) 370,256-268). Since GTP competes for initiation with the initiatingoligonucleotide, using a low GTP concentration favors GpA initiation butresults in low transcription yield (maximum calculated yield estimatedto be <150 ug/mL of reaction). When initiating transcription on “CT”template with ApG, CpG, UpG or GpG, they observed formation of RNAtranscripts with an additional untemplated 5′ nucleotide (A, C, U or G,respectively).

Axelrod et al. also used uncapped GpG dinucleotide to initiate RNAsynthesis on a promoter where template nucleotides +1 and +2 were2′-deoxycytidines (“CC” template). They observed low fidelity ofinitiation and observed three different transcription products. Theystate, “An examination of the autoradiograph indicates that one memberof each triplet resulted from initiation with GpG at the normal (+1)position, the second member of each triplet resulted from initiationwith GpG at the abnormal (−1) position, and the third member of eachtriplet resulted from initiation with guanosine triphosphate at thenormal position. Thus, GpG is a relatively weak initiator with the ø 10promoter (“CC” template), as well as with the ø 1.1 A promoter (“CT”template), and is unable to prevent normal initiation with guanosinetriphosphate at the concentration that was used.” They did not observeinitiation with a GpA dinucleotide with ø 10 promoter (“CC” template).CpA, ApC and ApA did not serve as initiators on either of the above “TC”and “CC” templates, presumably because these cannot hybridize to thetemplate nucleotides at positions +1 and +2. The method described inAxelrod et al. was designed for production of very small amounts ofradioactive transcripts for sequencing and is not suitable for largescale production of useful pharmaceutical amounts of RNA. If largerconcentrations (˜5 mM) of initiating dimer and NTPs, including GTP, wereused to increase the yield of RNA, the expected result is a lowproportion of RNA that starts with initiating dimer since GTP competesefficiently with dimer for initiation from the +1 nucleotides at NTPconcentrations closer to the Kd (2 mM), producing large proportion ofRNA that starts with pppG.

Pitulle et al. showed that transcription with T7 RNAP could be initiatedwith uncapped oligonucleotides (2-mer to 6-mer) (Gene, 112:101-105(1992)). These oligonucleotides had either a 5′-OH or a 5′monophosphate. They also initiated transcription with an oligonucleotideof the structure Biotin-ApG. All of the oligonucleotides used in thisstudy contained a 3′ terminal G. Pitulle et al. also showed that2′-O-methyl residues and deoxy residues could be included within theprimer sequence to produce RNA transcripts with 2′-O-methylated or2′-deoxy residues at or near 5′-end of RNA. It is clear in thispublication, that the 3′ terminal guanosine residue of the anyinitiating oligonucleotide paired with the +1 template nucleotide. Thisresults in untemplated nucleotides appended to the 5′ end of thetranscribed RNA. Specifically, the authors state, “Also sequencevariations in this segment are easily possible, since no base-pairingwith the template DNA is required apart from the Y-terminal G.” Thusnone of the initiating oligonucleotide primers is completelycomplementary only to the template nucleotide “C” at position +1 and notto any nucleotides at the following positions (+2, +3 etc.). This isconfirmed in subsequent methods papers by this group (Methods Mol Biol.74: 99-110 (1997), Methods Mol. Biol. 252:9-17, (2004)). Their methodsdiffer from the method described herein where all the nucleotides of theinitiating capped oligonucleotide primer completely complementary totemplate nucleotides at positions +1 and following positions. Kleineidamet al. created 5′ modified tRNA transcripts by initiation with dimers ortrimers that were modified with 2′-deoxy or 2′-O-methyl sugars (NucleicAcids Research 21:1097-1101 (1993). Again the authors state that the3′-terminal guanosine of the primer initiates at the +1 templatenucleotide “C”.

Another study by Ishikawa et al. showed that capped initiatingoligonucleotide trimers of the structure ^(m7)GpppApG,^(m7)Gppp^(m6)ApG, ^(m7)GpppA_(2′Ome)pG or ^(m7)Gppp^(m6)A_(2′Ome)pGcould initiate transcription on template with 2′-deoxycytidine residuesat template positions +1 and +2 (“CC” template; Nucleic Acids SymposiumSeries No. 53: 129 (2009)). The authors state, “The different resultfrom the case of using ^(m7)G5′pppG may be caused from base pairingbetween additional adenosine (N1) in ^(m7)G5′pppN1pG and2′-deoxythymidine in T7 promoter at −1 position.” This method clearlydiffers from the method described in the present invention where the +1and +2 nucleotides of the initiating capped oligonucleotide trimer pairwith the +1 and +2 of the template nucleotides. Ishikawa et al. used 6mM initiating oligonucleotide trimer, 0.9 mM GTP and 7.5 mM each of ATP,CTP and UTP. The authors used a greater than 6 fold excess of the cappedinitiating oligonucleotide primer, the most expensive nucleotidecomponent of transcription reaction, over competing GTP to drive thetranscription reaction toward capped RNA over pppRNA which increases thetotal cost of synthesized RNA. On the other hand, a low concentration ofGTP (0.9 mM) limits the total yield of the RNA in transcription reaction(theoretically to less then 1.4 mg/mL). On the contrary the methoddescribed herein does not require restricting the concentration of anyNTP to achieve both an efficient RNA capping and a higher yield of RNA(2 to 6 mg/mL) and thus allowing a production of high quality mRNA at acommercially useful cost.

None of the publications discussed above directly measured RNA cappingefficiency so the extent of capping in those studies is unknown.

Importantly, in all studies described above, the +1 template nucleotideis 2′-deoxycytidine (Biochemistry 24:5716-5723 (1985), Gene, 112:101-105(1992), Methods Mol. Biol. 74: 99-110 (1997), Methods Mol. Biol.252:9-17, (2004), Nucleic Acids Research 21:1097-1101 (1993), NucleicAcids Symposium Series No. 53: 129 (2009)).

In more than 20 years since the publication of the transcriptioninitiation studies with oligonucleotide primers, there have been nopublished examples of transcription initiation with initiatingoligonucleotide primers containing 5′- to 5′ inverted cap structuresuntil a publication of short report in Nucleic Acids Symposium SeriesNo. 53: 129 (2009).

The methods and compositions provided herein for preparation of5′-capped RNA include, but are not limited to, mRNA, small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), small cajal body-specific RNA(scaRNA). These methods involve the use of a Cap containingoligonucleotide primers, nucleoside 5′-triphosphates (NTPs) and RNApolymerase for DNA-templated and promoter controlled synthesis of RNA.In certain aspects, the methods use an initiating capped oligonucleotideprimer that provides utility in RNA synthesis, in particular synthesisof capped mRNAs. The initiating oligonucleotide primer has a structurethat resembles Cap 0, Cap 1, Cap 2 or TMG-Cap of natural RNA molecules,which include 2′-O-methylated nucleoside units at, penultimate Cap 1 andnext to penultimate Cap 2 5′-positions of RNA. The natural Cap 0structure does not have 2′-O-methylated nucleoside units.

The methods and compositions for preparation of RNA including, but notlimited to, mRNA, snRNA, snoRNA, scaRNA, transfer RNA (tRNA), ribosomalRNA (rRNA), and transfer-messenger RNA (tmRNA) that carry modificationsat or near 5′-end of the molecule. These methods involve the use ofinitiating oligonucleotide primers with or without Cap, nucleoside5′-triphosphates (NTPs) and RNA polymerase for DNA-templated andpromoter controlled synthesis of RNA. In certain aspects, the methodsuse a modified initiating oligonucleotide primer carrying structuralmodifications that provide utility in RNA synthesis; in particularsynthesis of 5′-modified RNAs.

The initiating capped oligonucleotide primer has an open 3′-OH groupthat allows for initiation of RNA polymerase mediated synthesis of RNAon a DNA template by adding nucleotide units to the 3′-end of theprimer. The initiating capped oligonucleotide primer is substantiallycomplementary to template DNA sequence at the transcription initiationsite (i.e., the initiation site is located closer to 3′-terminus of apromoter sequence and may overlap with promoter sequence). In certainembodiments, the initiating capped oligonucleotide primer directssynthesis of RNA predominantly in one direction (“forward”) startingfrom the 3′-end of the primer. In certain aspects and embodiments, theinitiating capped oligonucleotide primer outcompetes any nucleoside5′-triphosphate for initiation of RNA synthesis, thereby maximizing theproduction of the RNA that starts with initiating capped oligonucleotideprimer and minimizing a production of RNA that starts with5′-triphosphate-nucleoside (typically GTP).

The manufacture of mRNA by in vitro transcription utilizes highly activephage RNA polymerases (T3, T7, SP6 and others). RNA polymerase worksunder control of specific promoter which is incorporated in DNA plasmidconstruct in front of a template nucleotide sequence. Transcriptionprocess usually starts with purine nucleoside 5′-triphosphate (typicallyGTP) and continues until the RNA polymerase encounters a terminatingsequence or completes the DNA template.

As discussed above, mCAP dinucleotide analogs, ^(7m)G(5′)ppp(5′) N, thatcontain Cap 0 have been used for initiation in vitro transcription(e.g., RNA 1: 957-967 (1995)). The capped RNA molecules produced usingthese dinucleotide analogs contain Cap 0. However only about 50% ofsynthesized capped RNA molecules have the correct “forward” orientationof Cap 0. To convert RNA with Cap 0 to RNA with Cap 1 an additionalenzymatic reaction must be preformed using (nucleoside-2′-O)methyltransferase. However this conversion may be not quantitative; itis not easy to control and it is difficult to separate the remaining Cap0 RNAs from Cap 1 RNAs. In addition, the competition from NTPs(specifically GTP) for initiation transcription further reduces thequantity of active capped RNA molecules produced.

In addition, modified dinucleotide analogs, such as^(7m)G_(3′Ome)(5′)ppp(5′) N and other related ARCA analogs, that carrymodified ^(7m)G residue with blocked 3′ and/or 2′ position on ribose,have been used for initiation of in vitro transcription (e.g., RNA7:1486-1495 (2001)). These ARCA cap analogs direct RNA synthesis only inthe “forward” orientation and therefore produce a RNA molecule with(natural) Cap 0 on the 5′-terminus (having 2′ and/or 3′ modifications to^(7m)G residue). Such RNAs are more active in translation systemscompared to RNAs prepared using standard dinucleotide analogs,^(7m)G(5′)ppp(5′) N. To convert RNA with ARCA Cap 0 to RNA with ARCA Cap1 an additional enzymatic reaction must be performed with(nucleoside-2′-O) methyltransferase similar to that required for thedinucleotide analogs previously discussed. This method has the samedisadvantages as that elaborated for the mCAP dinucleotide analogs; theconversion of RNA with Cap 0 to RNA with ARCA Cap 1 may be notquantitative; the reaction is not easy to control, it is difficult toseparate remaining Cap 0 RNAs from Cap 1 RNAs and competition from NTPs(specifically GTP) for initiation of transcription further reduces thequantity of active capped RNA molecules produced.

Short oligonucleotide primers (2 to 6-mer) with 3′-terminal guanosineresidue have been used for initiation of in vitro transcription(Pitulle, C. et al., Gene, 112:101-105(1992)). These oligonucleotideprimers contained modified and unmodified ribonucleoside residues (e.g.,modified ribonucleoside residues included 2′-O-methylated nucleosideresidues and 2′-deoxyribonucleoside residues). The shorteroligonucleotide primers (dimers to tetramer) substantially out-competeGTP for initiation of transcription while longer primers (pentamer tohexamer) are much less efficient in initiation of transcription comparedto GTP. It may be because these longer primers (as they are designed)have a low percent of complementarity with DNA template at initiationsite. In contrast, dimer, AG (as designed), was complementary to the DNAtemplate at initiation site. The RNA molecules produced usingoligonucleotide primers, discussed in this section, had internal2′-O-methylated nucleoside but did not contain 5′-Cap 0, Cap1, Cap 2 orTMG-cap. To convert RNA without cap structure to RNA with Cap 1, Cap 2or TMG-cap structure an additional enzymatic reactions using cappingenzymes would have to be performed. However such conversion has the samedisadvantages as those stated above.

Other short RNA oligonucleotides containing cap structures and internal2′-O-methylated nucleoside residues have been chemically prepared(Ohkubo et al., Org. Letters 15:4386-4389 (2013)). These short cappedoligonucleotides were ligated with a “decapitated” (without 5′-capstructure) fragment of long RNA using T4 DNA ligase and a complementaryDNA splinter oligonucleotode. The final RNA synthesized using thischemical-enzymatic method had both internal 2′-O-methylated nucleosideresidues and 5′-TMG-cap structure. However only short capped RNAs(<200-mer) were prepared using this ligation approach. Moreover, theyields were low (15-30%). It is not easy to control and optimize the T4DNA ligation reaction and it requires a laborious separation processusing PolyAcrylamide Gel Electrophoresis and isolation of capped RNAsfrom remaining uncapped RNAs. Separation of long (500-10000 bases)capped mRNAs from remaining uncapped mRNAs by PAGE method is notfeasible.

Finally, 5′-modified nucleoside or 5′-modified mononucleotide or5′-modified dinucleotide, typically a derivative of guanosine, have beenused for initiating in vitro transcription of RNA (Gene, 112:101-105(1992) and Bioconjug. Chem., 10371-378 (1999)). These initiatornucleosides and nucleotides may carry labels or affinity groups (e.g.biotin) and, when incorporated on the 5′-end of RNA, would allow foreasy detection, isolation and purification of synthesized RNA. This5′labeled or tagged RNAs may be necessary for some applications. Howeverthis strategy was not used for the preparation of mRNA with Cap 0, Cap1, Cap 2 or TMG-cap structures.

In certain aspects of the present invention, compositions of theinitiating capped oligonucleotide primers of Formula I are provided. Inrelated aspects, are methods in which RNA is synthesized using theinitiating capped oligonucleotide primers of Formulas I.

Initiating Capped Oligonucleotide Primer

The initiating capped oligonucleotide primers of the present inventionhave a hybridization sequence which may be complementary to a sequenceon DNA template at initiation site. The length of the hybridizationsequence of the primers for use in the methods and compositions providedherein depends on several factors including the identity of templatenucleotide sequence and the temperature at which this primer ishybridized to DNA template or used during in vitro transcription.Determination of the desired length of a specific nucleotide sequence ofan initiating capped oligonucleotide primer for use in transcription canbe easily determined by a person of ordinary skill in the art or byroutine experimentation. For example, the length of a nucleic acid oroligonucleotide may be determined based on a desired hybridizationspecificity or selectivity.

In some embodiments, the nucleotide length of initiating cappedoligonucleotide primer (including the inverted 5′-5′ Cap nucleotide) isbetween 3 to about 9, in some embodiments the nucleotide length ofinitiating capped oligonucleotide primer (including Cap) is between 3 toabout 7, in some embodiments the nucleotide length of initiating cappedoligonucleotide primer (including Cap) is between 3 to about 5, and insome embodiments the nucleotide length of initiating cappedoligonucleotide primer (including Cap) is about 3. The length ofhybridization sequence within the initiating capped oligonucleotideprimer may be equal to or shorter than the total length of initiatingcapped oligonucleotide primer.

The presence of hybridization sequence forces an initiating cappedoligonucleotide primer to predominantly align with complementarysequence of the DNA template at the initiation site in only the desiredorientation (i.e., the “forward” orientation). In the forwardorientation, the RNA transcript begins with the inverted guanosineresidue (i.e., ^(7m)G(5′)ppp(5′) N . . . ) The dominance of the forwardorientation of the primer alignment on DNA template (FIG. 1) overincorrect “reverse” orientation is maintained by the thermodynamics ofthe hybridization complex. The latter is determined by the length of thehybridization sequence of initiating capped oligonucleotide primer andthe identity of bases involved in hybridization with DNA template.Hybridization in the desired forward orientation may also depend on thetemperature and reaction conditions at which DNA template and initiatingcapped oligonucleotide primer are hybridized or used during in vitrotranscription.

The initiating capped oligonucleotide primer of the present inventionenhances efficacy of initiation of transcription compared to efficacy ofinitiation with standard GTP, ATP, CTP or UTP. In some embodiments,initiation of transcription is considered enhanced when synthesis of RNAstarts predominantly from initiating capped oligonucleotide primer andnot from any NTP in transcription mixture. The enhanced efficiency ofinitiation of transcription results in a higher yield of RNA transcript.The enhanced efficiency of initiation of transcription may be increasedto about 10%, about 20%, about 40%, about 60%, about 80%, about 90%,about 100%, about 150%, about 200% or about 500% over synthesis of RNAwith conventional methods without initiating capped primer. In certainembodiments “initiating capped oligonucleotide primers” out-compete anyNTP (including GTP) for initiation of transcription. One of ordinaryskill in the art is able to readily determine the level of substrateactivity and efficacy of initiating capped oligonucleotide primers. Oneexample of a method of determining substrate efficacy is illustrated inExample 13). In certain embodiments, initiation takes place from thecapped oligonucleotide primer rather than an NTP, which results in ahigher level of capping of the transcribed mRNA.

In some aspects, methods are provided in which RNA is synthesizedutilizing an initiating capped oligonucleotide primer that hassubstitutions or modifications. In some aspects, the substitutions andmodifications of the initiating capped oligonucleotide primer do notsubstantially impair the synthesis of RNA. Routine test syntheses can bepreformed to determine if desirable synthesis results can be obtainedwith the modified initiating capped oligonucleotide primers. Thoseskilled in the art can perform such routine experimentation to determineif desirable results can be obtained. The substitution or modificationof initiating capped oligonucleotide primer include for example, one ormore modified nucleoside bases, one or more modified sugars, one or moremodified internucleotide linkage and/or one or more modifiedtriphosphate bridges.

The modified initiating capped oligonucleotide primer, which may includeone or more modification groups of the methods and compositions providedherein, can be elongated by RNA polymerase on DNA template byincorporation of NTP onto open 3′-OH group. The initiating cappedoligonucleotide primer may include natural RNA and DNA nucleosides,modified nucleosides or nucleoside analogs. The initiating cappedoligonucleotide primer may contain natural internucleotidephosphodiester linkages or modifications thereof, or combinationthereof.

In one embodiment the modification group may be a thermally labile groupwhich dissociates from a modified initiating capped oligonucleotideprimer at an increasing rate as the temperature of the enzyme reactionmedium is raised. Examples of thermally labile groups foroligonucleotides and NTPs are described in Nucleic Acids Res., 36:e131(2008), Collect. Symp. Ser., 10:259-263 (2008) and Analytical Chemistry,81:4955-4962 (2009).

In some aspects, methods are provided in which RNA is synthesized whereat least one or more NTP is added to a transcription reaction may have amodification as disclosed herein. In some aspects, the modification ofthe at least one NTP does not substantially impair RNA polymerasemediated synthesis of RNA. The modification of NTP may include forexample, one or more modified nucleoside bases, one or more modifiedsugars, one or more modified 5′-triphosphate. The modified NTP mayincorporate onto the 3′-end of the initiating capped oligonucleotideprimer and it does not block transcription and supports furtherelongation of the primer.

In another embodiment, the modification group of an initiating cappedoligonucleotide primer may be a detectable label or detectable marker.Thus, following transcription, the target RNA, containing the detectablelabel or marker, can be identified by size, mass, color and/or affinitycapture. In some embodiments, the detectable label or marker is afluorescent dye; and the affinity capture label is biotin. In certainembodiments, one or more components of a transcription reaction(initiating capped oligonucleotide primer and/or NTPs) may be labeledwith a detectable label or marker. Thus, following transcription, theRNA molecule can be identified, for example, by size, mass, affinitycapture or color. In some embodiments, the detectable label is afluorescent dye; and the affinity capture label is biotin.

Standard chemical and enzymatic synthesis methods may be utilized tosynthesize the “initiating capped oligonucleotide primers” of thepresent invention and are disclosed herein in the Examples section.

Kits

Kits including, the “initiating capped oligonucleotide primer” forperforming transcription are also contemplated. For example, kits maycontain all transcription reagents for synthesis of common RNAs (e.g.,FLuc mRNA). More specifically, a kit may contain: an “initiating cappedoligonucleotide primer”; a container marked for transcription;instructions for performing RNA synthesis; and one or more reagentsselected from the group consisting of one or more modified or unmodifiedinitiating capped oligonucleotide primers, one or more unmodified NTPs,one or more modified NTPs (e.g., pseudouridine 5′-triphosphate), an RNApolymerase, other enzymes, a reaction buffer, magnesium and a DNAtemplate.

The initiating capped oligonucleotide primers of the present inventionhave a significant advantage over current methods and compositionsinvolving use of various initiating nucleosides, nucleotides andoligonucleotides or use of polyphosphate dinucleotide derivativescontaining Cap 0 structure, such as mCAP and ARCA. The initiating cappedoligonucleotide primers are compatible with existing transcriptionsystems and reagents and no additional enzymes or reagents are required.In addition, the use of initiating capped oligonucleotide primers makesseveral non-enzymatic and enzymatic steps (such as capping and2′-O-methylation) unnecessary thus reducing complexity of the processand a cost of RNA synthesis.

While the exemplary methods described herein relate to T7 RNA polymerasemediated transcription reaction, a number of other RNA polymerases knownin the art for use in transcription reactions may be utilized with thecompositions and methods of the present invention. Other enzymes,including natural or mutated variants that may be utilized include, forexample, SP6 and T3 RNA polymerases and RNA polymerases from othersources including thermostable RNA polymerases.

Some nucleic acid replication and amplification methods may includetranscription as a part of the process. Among these methods are:transcription mediated amplification (TMA) and nucleic acidsequence-based amplification (NASBA), DNA and RNA sequencing and othernucleic acid extension reactions known in the art. The skilled artisanwill understand that other methods may be used either in place of, ortogether with, transcription methods, including variants oftranscription reactions developed in the future.

Therapeutic Uses

The present invention also contemplates the production of mRNAscontaining the initiation capped oligonucleotide primer for use astherapeutic agents in a pharmaceutical composition, the introduction ofRNAs containing the initiating capped oligonucleotide primer into cellsto treat a medical condition of the cells or the introduction of RNAscontaining the initiating capped oligonucleotide primer into cells thatutilize those RNAs to produce proteins that may have a therapeuticaffect on the host cells.

One method for treating a condition utilizing an RNA containing aninitiating capped oligonucleotide primer comprises the step ofadministering the RNA containing the initiating capped oligonucleotideprimer of formula I or a composition comprising such RNA to a subjecthaving, or suspected of having a condition whose symptoms/symptomologiesmay be reduced in severity or eliminated.

An RNA containing the initiating capped oligonucleotide primer offormula I “compound”, when formulated in a pharmaceutically acceptablecarrier and/or pharmaceutically acceptable salt at a concentration of 4mg/ml or less, is effective to produce a reduction of the symptomsand/or symptomologies by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or greater than an untreated individual with the pharmaceuticallyacceptable carrier alone.

Pharmaceutical compositions may be formulated for administration byinjection or other appropriate routes known to those skilled in the artfor treating a particular condition. An injectable composition forparenteral administration typically contains the active compound in asuitable solution and/or pharmaceutical carrier such as sterilephysiological saline. The composition may also be formulated as asuspension in a lipid or phospholipid, in a liposomal suspension, or inan aqueous emulsion.

Methods for preparing a variety of compositions and/or formulations areknown to those skilled in the art see Remington's PharmaceuticalSciences (19^(th) Ed., Williams & Wilkins, 1995). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically safe and effective amount for increasing expression ofthe desired protein in the target cells or tissue.

In some embodiments, the pharmaceutical composition contains at least0.1% (w/v) of the compound, as described above, in some embodiments, thepharmaceutical composition contains greater than 0.1%, in someembodiments, the pharmaceutical composition contains up to about 10%, insome embodiments, the pharmaceutical composition contains up to about5%, and in some embodiments, the pharmaceutical composition contains upto about 1% (w/v) of the compound. Choice of a suitable concentrationdepends on factors such as the desired dose, frequency and method ofdelivery of the active agent.

For treatment of a subject, such as a mammal or a human, dosages aredetermined based on factors such as the weight and overall health of thesubject, the condition treated, severity of symptoms, etc. Dosages andconcentrations are determined to produce the desired benefit whileavoiding any undesirable side effects. Typical dosages of the subjectcompounds are in the range of about 0.0005 to 500 mg/day for a humanpatient, and ranging in some embodiments between about 1-100 mg/day. Forexample, higher dose regimens include e.g. 50-100, 75-100, or 50-75mg/day, and lower dose regimens include e.g. 1-50, 25-50, or 1-25mg/day.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.One of ordinary skill in the art readily knows how to synthesize orcommercially obtain the reagents and components described herein.

Example 1 Preparation of 7-methylguanosine 5-diphosphate (pp^(7m)G) fromguanosine 5′-diphosphate FIG. 1

To a stirring solution of guanosine 5′-diphosphate (2.5 mmol) in 40.0 mLof water, acetic acid is added to adjust the pH of the solution to 4.0.To this mixture dimethyl sulfate (4.0 mL) is added dropwise over aperiod of 30 minutes and the reaction mixture is stirred at roomtemperature for 4 hours while maintaining the pH of the reaction mixtureat about 4.0 using 0.1M NaOH solution. After 4 hours, the reactionmixture is extracted with CH₂Cl₂ (3×50 mL) to remove unreacted dimethylsulfate. The aqueous layer is diluted with water to 500 mL, adjusted topH 6.5 with 1M TEAB and loaded on a DEAE Sephadex column (3×50 cm). Theproduct is eluted using a linear gradient of 0-1 M TEAB, pH 7.5 (3 L).Fractions containing pure pp^(7m)G (triethylammonium salt) are pooled,evaporated, and dried under high vacuum to give a fine white powder(yield: 80%). A similar procedure is disclosed in Bioorgan. Med. Chem.Letters 17:5295-5299 (2007).

Example 2 Preparation of 7-methylguanosine 5′-diphosphate imidazolide(Im-pp^(7m)G) from pp^(7m)G FIG. 2

The triethylammonium salt of pp^(7m)G (0.4 mmol) is reacted withimidazole (4 mmol), triphenylphosphine (2 mmol) and 2, 2′-dipyridyldisulfide (2 mmol) in dry DMF (20 mL) for 8 hrs. Crude Im-pp^(7m)G isprecipitated by pouring the reaction mixture into 250 mL of a 0.2 Msodium perchlorate solution. The mixture is cooled to −20° C. and theresulting precipitate is collected by centrifugation, washed withacetone (3×50 mL) and dried under high vacuum. The isolated yield ofIm-pp^(7m)G is approximately 100%. A similar procedure is disclosed inNucleosides, Nucleotides, and Nucleic Acids 24:1131-1134 (2005) and J.Org. Chem. 64:5836-5840 (1999).

Example 3 Preparation of 3′-O-Methylguanosine 5′-phosphate(pG(_(3′Ome))) from 3′-O-methylguanosine FIG. 3

3′-O-methylguanosine (10 mmol) is dissolved in triethylphosphate (40 mL)at 60-70° C. The mixture is cooled to 0° C. in an ice-water bath,phosphorus oxychloride (30 mmol) is added and the mixture is stirredunder argon for 3 hrs at room temperature. The reaction is quenched byslow addition of 1M TEAB (100 mL; pH 8.5) with stirring. The mixture isstirred for 8 hrs and diluted with 1 L of water. The resulting solutionis loaded onto a DEAE Sephadex column (3×40 cm) and the product iseluted with a linear gradient 0.05 to 1.0M (3 L) of TEAB (pH 7.5).Fractions containing pure product are combined, evaporated to a solidresidue and co-evaporated with methanol (4×50 mL) to give pG(_(3′Ome))(triethylammonium salt) as a white solid (yield 60%). A similarprocedure is disclosed in U.S. Patent Application serial no.2012/0156751.

Example 4 Preparation of 3′-O-Methylguanosine 5′-phosphorimidazolide(Im-pG(_(3′Ome))) from pG(_(3′Ome)) FIG. 4

The triethylammonium salt of pG(_(3′Ome))(0.5 mmol) is reacted withimidazole (5 mmol), triphenylphosphine (2.5 mmol) and 2,2′-dipyridyldisulfide (2.5 mmol) in dry DMF (25 mL) for 5 hrs. Crude Im-pG(_(3′Ome))is precipitated by pouring the reaction mixture into 400 mL of 0.2 Msodium perchlorate in acetone solution. The mixture is cooled to −20° C.and the resulting precipitate is collected by centrifugation, washedwith acetone (3×60 mL) and dried under high vacuum (yield: 100%). Asimilar procedure is disclosed in U.S. Patent Application serial no.2012/0156751.

Example 5 Preparation of 3′-O-Methylguanosine 5′-diphosphate(ppG(_(3′Ome))) from Im-pG(_(3′Ome)) FIG. 5

Solid zinc chloride (14.0 mmol) is added with small portions to asolution of Im-pG(_(3′Ome)) (7.0 mmol) in dry DMF (40 mL). The mixtureis stirred for 15 minutes under argon until all solids are dissolved. Asolution of 1M tributylammonium phosphate in DMF (40 mL) is added andthe mixture is stirred at room temperature. After 5 hours the mixture isdiluted with 200 mL of water and extracted with dichloromethane (2×200mL). The aqueous layer is diluted with water (1 L), loaded onto a DEAESephadex column (5×40 cm) and eluted with linear gradient of 0.05 to1.0M (6 L) TEAB (pH 7.5). Fractions containing pure product arecombined, evaporated and co-evaporated with methanol (4×50 mL) to giveppG(_(3′Ome)) (triethylammonium salt) as a white solid (yield: 60%). Asimilar procedure is disclosed in U.S. Patent Application serial no.2012/0156751.

Example 6 Preparation of 7-methyl-3′-O-methylguanosine 5-diphosphate(pp^(7m)G(_(3′Ome))) from ppG(_(3′Ome)) FIG. 6

A solution of ppG(_(3′Ome)) (triethylammonium salt; 3.0 mmol) in 50 mLof water is prepared and glacial acetic acid is added to adjust the pHof the solution to 4.0. Dimethyl sulfate (10.0 mL) is added dropwise tothis mixture over a period of 30 minutes and the reaction mixture isstirred at room temperature for 4 hours while maintaining a pH of4.0±0.5 using 0.1M NaOH solution. After 4 hours, the reaction mixture isextracted with CH₂Cl₂ (3×150 mL) to remove unreacted dimethyl sulfate.The aqueous layer is adjusted to pH 5.5, diluted with water (500 mL) andloaded onto a DEAE Sephadex column (3×50 cm). The product is elutedusing a linear gradient of 0-1.0M TEAB, pH 7.5 (3 L). Fractionscontaining pure pp^(7m)G_(3′Ome) (triethylammonium salt) are pooled,evaporated, and dried under high vacuum to give a fine white powder(yield: 80%). A similar procedure is disclosed in RNA 9:1108-1122(2003);Nucleoside Nucleotides & Nucleic acids 25:337-340 (2006); and U.S.Patent Application Serial no. 2012/0156751.

Example 7 Preparation of 7-methyl-3′-O-methylguanosine 5-diphosphateimidazolide (Im-pp^(7m)G(_(3′Ome))) from pp^(7m)G(_(3′Ome)) FIG. 7

The protocol described in Example 1 is utilized for preparation ofIm-pp^(7m)G(_(3′Ome)). A similar procedure is disclosed in RNA14:1119-1131 (2008).

Example 8 General Procedure for Preparation of pN(_(2′-OR1))pNDinucleotides (R₁═H or Me) FIG. 8

Phosphoramidite monomer (i) (1.0 mmol) and 2′,3′, N-protected nucleoside(ii) (1.0 mmol) are reacted in 10 mL of acetonitrile containing 2.5molar equivalents of activator (tetrazole). After 60 minutes of stirringat room temperature the intermediate product is oxidized from the P(III)to P(V) state with iodine and extracted with dichloromethane (200 mL)and brine (200 mL). The organic layer is dried with sodium sulfate andis evaporated to solid foam (intermediate (iii)).

To remove the DMT-protecting group, intermediate (iii) is dissolved in10 mL of 80% acetic acid and, after reaction is completed (about 1-2hrs), the mixture is evaporated and co-evaporated with methanol (5×30mL) to remove acetic acid. The crude 5′-OH dimer (iv) is isolated andpurified by silica gel chromatography using 5% methanol indichloromethane as an eluent.

The 5′-OH dimer (iv) (1.0 mmol) is phosphitylated with 2 equivalents ofbis-cyanoethyl-N,N-diisopropyl-phosphoramidite and 2 equivalents ofactivator (tetrazole) in 10 mL of acetonitrile. After 30 minutes ofstirring at room temperature the 5′-phosphitylated dimer is oxidizedfrom the P(III) to P(V) state with iodine and extracted withdichloromethane (150 mL) and brine (150 mL). The organic layer isevaporated to an oily residue, co-evaporated with methanol (2×30 mL),dissolved in 12 mL of methanol and concentrated ammonia (12 mL) wasadded. The mixture is kept at room temperature for over 48 hours untildeprotection of the pN_(2′OR1)pN dimer (v) is complete. The mixture isevaporated and co-evaporated with methanol (2×30 mL).

When R=methyl the crude dimer (v) is directly purified by anion exchangeand reverse phase chromatography (Step 5). Fractions are evaporated togive a final pN_(2′OR1)pN dimer (v) (v; R₁=methyl; triethylammoniumsalt) as a white solid (35% overall yield).

When R=TBDMS the crude dimer (v) is treated (Step 4B) with HF-3TEAmixture to remove 2′-OTBDMS protecting group (Org. Biomol. Chem.3:3851-3868 (2005) and Nucl. Acids Res. 22:2430-2431 (1994)). When thereaction is complete, the mixture is diluted with 0.05M TEAB andpurified by anion exchange and reverse phase chromatography (Step 5).Fractions are evaporated to give a final pN_(2′OR1)pN dimer (v) (R₁═H;triethylammonium salt) as a white solid (30% overall yield).

Example 9 General Procedure for the Synthesis of InitiatingOligonucleotides with Cap 0, Cap 1 or Cap 2 Structures(5′-phosphorylated Dinucleotide is Used in Example) FIG. 9

A. Approach 1:

To a suspension of Im-pp^(7m)G(_(3′Ome)) or Im-pp^(7m)G (2 mmol; sodiumsalt form) and 5′-phosphorylated dinucleotide (1 mmol; triethylammoniumsalt) in DMF (50 mL), anhydrous ZnCl₂ (1 g) is added slowly while themixture is stirred at 35° C. After 24 hours the reaction is stopped byaddition of a 25 mM solution of EDTA in water (500 mL) and neutralizedby 1M solution of sodium bicarbonate. The mixture is diluted to 1 L withwater and loaded on a DEAE Sephadex column (3×50 cm). The product iseluted using a linear gradient of 0-1 M ammonium bicarbonate, pH 7.2 (2L). Fractions containing pure product are pooled, evaporated, and driedunder high vacuum to give a fine white powder (yield: 60%). A similarapproach is disclosed in U.S. patent application serial no.2012/0156751; Bioorg. Med. Chem. Lett. 17:5295-5299 (2007) and RNA14:1119-1131 (2008).

B. Approach 2:

Im-pp^(7m)G(_(3′Ome)) or Im-pp^(7m)G (2 mmol) is dissolved in N-methylmorpholine buffer (0.2 M, pH 7.0, 10 mL) containing MnCl₂ (2 mmol) andadded to solid 5′-phosphorylated dinucleotide (1 mmol; triethylammoniumsalt). The reaction is stirred at room temperature. After 24-40 hoursthe reaction is stopped with 10 mL of 0.25M solution of EDTA. Themixture is loaded onto a DEAE Sephadex column (3×50 cm). The product iseluted using a linear gradient of 0-1.0M ammonium bicarbonate, pH 7.2 (2L). Fractions containing pure product are pooled, evaporated, and driedunder high vacuum to give a fine white powder (yield 50%). A similarapproach is disclosed in Bioorganic & Medicinal Chemistry 21:7921-7928(2013), Nucleic Acids Research 37:1925-1935 (2009); J. Org. Chem.64:5836-5840 (1999). (Note: 1. A 5′-phosphorylated trimer, tetramer,pentamer, hexamer, heptamer, octamer, nanomer or decamer oligonucleotidecan be used instead of 5′-phosphorylated dinucleotide as illustrated inExample 9. 2. In order to prepare the initiating capped oligonucleotidewith Cap 0, the 5′-phosphorylated oligonucleotide does not have2′-O-methyl groups on the first 5′-nucleoside residues, e.g. pApG. 3. Inorder to prepare the initiating capped oligonucleotide with Cap 1, the5′-phosphorylated oligonucleotide carries one 2′-O-methyl group on thefirst 5′-nucleoside residue, e.g. pA(_(2′Ome))pG. 4. In order to preparethe initiating capped oligonucleotide with Cap 2, the 5′-phosphorylatedoligonucleotide carries two 2′-O-methyl groups on the first and second5′-nucleoside residues, e.g. pA(_(2′Ome))pG(_(2′Ome))pG).

Example 10 Structures of Initiating Capped Oligonucleotide PrimersAccording to Formula I

FIG. 10 shows the structures of the initiating capped oligonucleotideprimers used in the examples according to Structure I. A)^(m7)G_(3′Ome)pppG, B) ^(m7)GpppG_(2′Ome)pG, C)^(m7)G_(3′Ome)pppG_(2′Ome)pG, D) ^(m7)GpppA_(2′Ome)pG, E)^(m7)G_(3′Ome)pppA_(2′Ome)pG, F) ^(m7)GpppC_(2′Ome)pG, G)^(m7)G_(3′Ome)pppC_(2′Ome)pG, H) ^(m7)GpppA_(2′Ome)pG_(2′Ome)pG.

Example 11 In Vitro Transcription with ARCA Primer

A double stranded DNA transcription template encoding firefly luciferasewas generated by polymerase chain reaction. The +1 template nucleotidewas 2′-deoxycytidine. Transcription reactions were assembled with 25ug/mL transcription template, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl₂, 2 mMspermidine, 10 mM DTT, 0.002% Triton X-100, 1000 unit/mL murine RNaseinhibitor (New England Biolabs catalog #M0314), 2 unit/mL inorganicpyrophosphatase (New England Biolabs catalog # M2403), 4000 units/mL T7RNA polymerase (New England Biolabs catalog #M0251), 6 mM ARCA(^(m7)G_(3′Ome)pppGG), 1.5 mM GTP, 7.5 mM ATP, 7.5 mM CTP and 7.5 mMUTP. Transcriptions were incubated at 37° C. for 2 hours. Reactions weresupplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl₂, 0.5 mM CaCl₂and 100 units/mL DNase I (New England Biolabs catalog #M0303) andincubated for 1 hour at 37° C. The resulting mRNAs were purified usingan RNeasy Maxi kit (Qiagen catalog #75162) according to manufacturer'sinstructions. mRNAs were eluted in water and dephosphorylated byadjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mMMgCl₂, 0.1 mM ZnCl₂ and 250 units/mg Antarctic phosphatase (New EnglandBiolabs catalog #M0289). The reaction were incubated at 37° C. for 1hour. The resulting mRNAs were purified using an RNeasy Maxi kit (Qiagencatalog #75162) according to manufacturer's instructions. mRNAs wereeluted in water.

Example 12 In Vitro Transcription with Trimeric Initiating CappedOligonucleotide Primers

Some initiating capped oligonucleotide primers that are utilized intranscriptions are indicated in Table 1. A double stranded fireflyluciferase DNA transcription template specific for each trimer isgenerated by polymerase chain reaction. These templates differed intemplate nucleotides +1 and +2. For a given trimer, template nucleotide+1 was complementary to B₁ (the nucleotide destined to become transcriptnucleotide +1). Likewise template nucleotide +2 was complementary to B₁₀(the nucleotide destined to become transcript nucleotide +2; see Example10)). Transcription reactions are assembled with 25 ug/mL transcriptiontemplate, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl₂, 2 mM spermidine, 10 mMDTT, 0.002% Triton X-100, 1000 unit/mL murine RNase inhibitor (NewEngland Biolabs catalog #M0314), 2 unit/mL inorganic pyrophosphatase(New England Biolabs catalog #M2403), 4000 units/mL T7 RNA polymerase(New England Biolabs catalog #M0251) and 6 mM of initiating cappedoligonucleotide primer, 1.5 mM GTP and 7.5 mM each of ATP, CTP and UTP.In subsequent examples, this primer/NTP formulation will be referred toas Primer/NTP Formulation 1. It is clear to one skilled in the art thatother polymerases such as T7, T3 or SP6 RNA polymerases could be usedinstead of T7 RNA polymerase to perform the same function by using theirrespective promoters. Transcription reaction mixtures are incubated at37° C. for 2 hours. Reactions are supplemented with 10 mM Tris-HCl (pH7.6), 2.5 mM MgCl₂, 0.5 mM CaCl₂) and 100 units/mL DNase I (New EnglandBiolabs catalog #M0303) and incubated for 1 hour at 37° C. The resultingmRNAs are purified using an RNeasy Maxi kit (Qiagen catalog #75162)according to manufacturer's instructions. mRNAs are eluted in water anddephosphorylated by adjusting the solution to 50 mM Bis-Tris-Propane HCl(pH 6.0), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 250 units/mg Antarcticphosphatase (New England Biolabs catalog #M0289). The reaction isincubated at 37° C. for 1 hour. The resulting mRNAs are purified usingan RNeasy Maxi kit (Qiagen catalog #75162) according to manufacturer'sinstructions. mRNAs are eluted in water.

Example 13 In Vitro transcription with Tetrameric Initiating CappedOligonucleotide Primer ^(7m)G_(3′Ome)pppA_(2′Ome)pG_(2′Ome)pG

Initiating capped oligonucleotide primer^(7m)G_(3′OMe)pppA_(2′OMe)pG_(2′OMe)pG were used in transcription.Transcription were carried out as in Example 11 with the followingmodifications. A double stranded DNA transcription template specific fortetramer was generated by polymerase chain reaction. The templatenucleotide +1 (2′-deoxythymidine) was complementary to Adenosine (thefirst nucleotide of the primer destined to become a transcriptnucleotide +1). Likewise template nucleotide +2 (2′-deoxycytidine) wascomplementary to guanosine (the second nucleotide of the primer destinedto become transcript nucleotide +2). Likewise template nucleotide +3(2′-deoxycytidine) was complementary to guanosine (the third nucleotideof the primer destined to become transcript nucleotide +3).Transcription reactions were assembled with 25 ug/mL transcriptiontemplate, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl₂, 2 mM spermidine, 10 mMDTT, 0.002% Triton X-100, 1000 unit/mL murine RNase inhibitor (NewEngland Biolabs catalog #M0314), 2 unit/mL inorganic pyrophosphatase(New England Biolabs catalog # M2403), 4000 units/mL T7 RNA polymerase(New England Biolabs catalog #M0251), 6 mM of initiating cappedoligonucleotide primer ^(7m)G_(3′OMe)pppA_(2′OMe)pG_(2′OMe)pG, 1.5 mM ofGTP, 7.5 mM of ATP, 7.5 mM of UTP and 7.5 mM of CTP. It is clear to oneskilled in the art that other polymerases such as T7, T3 or SP6 RNApolymerases could be used instead of T7 RNA polymerase to perform thesame function by using their respective promoters. Transcriptionreaction mixtures were incubated at 37° C. for 2 hours. Reactions weresupplemented with 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl₂, 0.5 mM CaCl₂and 100 units/mL DNase I (New England Biolabs catalog #M0303) andincubated for 1 hour at 37° C. The resulting mRNAs were purified usingan RNeasy Maxi kit (Qiagen catalog #75162) according to manufacturer'sinstructions. mRNAs were eluted in water and dephosphorylated byadjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mMMgCl₂, 0.1 mM ZnCl₂ and 250 units/mg Antarctic phosphatase (New EnglandBiolabs catalog #M0289). The reaction was incubated at 37° C. for 1hour. The resulting mRNAs were purified using an RNeasy Maxi kit (Qiagencatalog #75162) according to manufacturer's instructions. mRNAs wereeluted in water.

Example 14 In Vitro Transcription with ^(m7)GpppA_(2′Ome)pG InitiatingCapped Oligonucleotide Primers on a Transcription Template with2′-deoxythymidine and 2′-deoxycytidine Residues at Template Positions +1and +2, Respectively

A double stranded firefly luciferase DNA transcription template was usedwith 2′-deoxythymidine and 2′-deoxycytidine residues at templatepositions +1 and +2, respectively. Two transcription reactions wereassembled with 25 ug/mL transcription template, 40 mM Tris-HCl (pH 8.0),27 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 0.002% Triton X-100, 1000unit/mL murine RNase inhibitor (New England Biolabs catalog #M0314), 2unit/mL inorganic pyrophosphatase (New England Biolabs catalog # M2403),4000 units/mL T7 RNA polymerase (New England Biolabs catalog #M0251).The two transcriptions differed in the amount of initiating cappedoligonucleotide, amount of NTPs and the identity of NTPs. The firsttranscription was designed to mimic the transcription conditions ofIshikawa et al. (Nucleic Acids Symposium Series No. 53:129 (2009)). Thistranscription reaction contained 6 mM of ^(m7)GpppA_(2′Ome)pG initiatingcapped oligonucleotide primer, 0.9 mM GTP and 7.5 mM of ATP, CTP andUTP. In subsequent examples this primer/NTP formulation will be referredto as Primer/NTP Formulation 2. In this formulation, the initiatingoligonucleotide primer was in greater than 6 fold excess over GTP. Thesecond transcription used 5 mM of ^(m7)GpppA_(2′Ome)pG initiating cappedoligonucleotide primer, 5 mM GTP, ATP, CTP and pseudouridinetriphosphate (PTP). In subsequent examples this primer/NTP formulationwill be referred to as Primer/NTP Formulation 3. In Primer/NTPFormulation 3, the GTP concentration was increased in order to producecommercially useful amounts of RNA. It is clear to one skilled in theart that other polymerases such as T7, T3 or SP6 RNA polymerases couldbe used instead of T7 RNA polymerase to perform the same function byusing their respective promoters. Transcription reaction mixtures areincubated at 37° C. for 2 hours. Reactions were supplemented with 10 mMTris-HCl (pH 7.6), 2.5 mM MgCl₂, 0.5 mM CaCl₂ and 100 units/mL DNase I(New England Biolabs catalog #M0303) and incubated for 1 hour at 37° C.The resulting mRNAs were purified using an RNeasy Maxi kit (Qiagencatalog #75162) according to manufacturer's instructions. mRNAs wereeluted in water and dephosphorylated by adjusting the solution to 50 mMBis-Tris-Propane HCl (pH 6.0), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 250 units/mgAntarctic phosphatase (New England Biolabs catalog #M0289). The reactionwas incubated at 37° C. for 1 hour for Primer/NTP Formulation 2 and 3hours for Primer/NTP Formulation 3 hour. The resulting mRNAs werepurified using an RNeasy Maxi kit (Qiagen catalog #75162) according tomanufacturer's instructions. mRNAs were eluted in water. The purifiedtranscription yields with Primer/NTP Formulation 2 and 3 were 0.7milligram/milliliter transcription reaction (mg/mL) and 3.9 mg/mLtranscription reaction, respectively. The yield with Primer/NTPFormulation 3 was greatly superior to that obtained with Primer/NTPFormulation 2.

Example 15 In Vitro Transcription with ^(m7)GpppA_(2′Ome)pG InitiatingCapped Oligonucleotide Primers on a Transcription Template with CytidineResidues at Template Positions +1 and +2

A double stranded firefly luciferase DNA transcription template was usedin which the +1 and +2 template nucleotides were cytidines and thus notcompletely complementary to the ^(m7)GpppA_(2′Ome)pG initiating cappedoligonucleotide primer. Two transcription reactions were assembled with25 ug/mL transcription template, 40 mM Tris-HCl (pH 8.0), 27 mM MgCl₂, 2mM spermidine, 10 mM DTT, 0.002% Triton X-100, 1000 unit/mL murine RNaseinhibitor (New England Biolabs catalog #M0314), 2 unit/mL inorganicpyrophosphatase (New England Biolabs catalog # M2403), 4000 units/mL T7RNA polymerase (New England Biolabs catalog #M0251). The twotranscriptions differed in the amount of initiating cappedoligonucleotide and NTPs. The first transcription was designed to mimicthe transcription conditions of Ishikawa et al. (Nucleic Acids SymposiumSeries No. 53: 129 (2009)). This transcription reaction containedPrimer/NTP Formulation 2. The second transcription used Primer/NTPFormulation 3. It is clear to one skilled in the art that otherpolymerases such as T7, T3 or SP6 RNA polymerases could be used insteadof T7 RNA polymerase to perform the same function by using theirrespective promoters. Transcription reaction mixtures were incubated at37° C. for 2 hours. Reactions were supplemented with 10 mM Tris-HCl (pH7.6), 2.5 mM MgCl₂, 0.5 mM CaCl₂ and 100 units/mL DNase I (New EnglandBiolabs catalog #M0303) and incubated for 1 hour at 37° C. The resultingmRNAs were purified using an RNeasy Maxi kit (Qiagen catalog #75162)according to manufacturer's instructions or by reverse phase highperformance liquid chromatography. mRNAs were dephosphorylated byadjusting the solution to 50 mM Bis-Tris-Propane HCl (pH 6.0), 1 mMMgCl₂, 0.1 mM ZnCl₂ and 250 units/mg Antarctic phosphatase (New EnglandBiolabs catalog #M0289). The reaction were incubated at 37° C. for 1hour for Primer/NTP Formulation 2 and 3 hours for Primer/NTP Formulation3. The resulting mRNAs were purified using an RNeasy Maxi kit (Qiagencatalog #75162) according to manufacturer's instructions. mRNAs wereeluted in water. The purified transcription yields with Primer/NTPFormulations 2 and 3 were 0.6 milligram/milliliter transcriptionreaction (mg/mL) and 3.9 mg/mL transcription reaction, respectively. Theyield with Primer/NTP Formulation 3 was greatly superior to thatobtained with Primer/NTP Formulation 2.

Example 16 Translation of mRNAs in Huh-7 Cells FIG. 11

Translational activity of luciferase mRNAs generated with initiatingcapped oligonucleotide primers was assessed in cultured hepatocytes intriplicate. Huh-7 cells were cultured in DMEM supplemented with 10% FBS,L-glutamine, non-essential amino acids and penicillin/streptomycin at37° C. under an atmosphere of 5% CO₂. Cells were transfected with 400 ngof mRNAs. For comparison, cells were also transfected with a Cap0luciferase mRNA generated by initiation with ARCA. At 20 hours, cellswere harvested and luciferase activity was measured using a ONE-GloLuciferase Assay System kit (Promega catalog #E6120) according tomanufacturer's recommendations. Luminescence was measured using aGloMax-Multi+Detection System instrument according to manufacturer'srecommendations. Luciferase activity was detected for all mRNAs tested(FIG. 11). The fact that mRNAs generated with initiating cappedoligonucleotide primers or 3′-O-methyl initiating capped oligonucleotideprimers translate with similar efficiencies to ARCA capped RNAsindicates that they are efficiently capped co-transcriptionally.

Example 17 Assay to Determine Capping Efficiency of mRNA Generated byCo-Transcriptional Capping with ARCA or Initiating CappedOligonucleotide Primer Using Primer/NTP Formulation 1 FIGS. 12A-12H

For each initiating capped oligonucleotide primer tested, sufficientquantities of mRNA to be detected by liquid chromatography massspectroscopy (LC-MS) was subjected to a capping assay. In this assay, asmall fragment was cleaved from the 5′ end of a full length mRNA andanalyzed by LC-MS. Prior to cleavage of the mRNA, it was treated withAntarctic phosphatase (New England Biolabs catalog # M0289) to convertuncapped monophosphates, diphosphates and triphosphates to a 5′ OH tofacilitate analysis. The phosphatase treated mRNA was then cleaved andpurified. The purified RNA was subjected to LC-MS analysis. FIG. 12shows the LC traces. LC peaks corresponding to uncapped (5′ OH afterphosphatase treatment) and Cap 1 are indicated with observed masses. Theinset schematic shows the alignment of the initiating oligonucleotideprimer on the transcription template. Note that “|” indicates a basepair of the capped initiating nucleotide with the template nucleotide inthe schematic. Subscript “m” indicates a 2′-O-methyl group andsuperscript “m7” indicates a base methylation. For comparison, an mRNAco-transcriptionally capped with ARCA was subjected to the cappingassay. An estimate of the capping efficiency was made using thefollowing formula (intensity of capped peaks)/[(intensity of cappedpeak)+(intensity of the 5′ OH peak)]. The observed % capping in FIG. 12was A) ^(m7)G_(3′Ome)pppG=79%, B) ^(m7)GpppG_(2′Ome)pG=89%, C)^(m7)G_(3′Ome)pppG_(2′Ome)pG=87%, D) ^(m7)GpppA_(2′Ome)pG=99%, E)^(m7)G_(3′Ome)pppA_(2′Ome)pG=99%, F) mGpppC_(2′Ome)pG=98%, G)^(m7)G_(3′Ome)pppC_(2′Ome)pG=97%, H) ^(m7)GpppA_(2′Ome)pG_(2′Ome)pG=50%.In each case, the capping efficiency of transcripts co-transcriptionallycapped with initiating trimeric capped oligonucleotide primers wasgreater than that observed with ^(m7)G_(3′Ome)pppG (ARCA).

Example 18 Comparison of Capping with ^(m7)GpppA_(2′,Ome)pG InitiatingCapped Oligonucleotide on a Transcription Template with2′-deoxythymidine and 2′-deoxycytidine Residues at Template Positions +1and +2 vs. A Transcription Template with Cytidine Residues at TemplatePositions +1 and +2 FIGS. 13A-13D

mRNAs made in Examples 14 and 15 were subjected to a capping assay todetermine relative efficiency and specificity of capping of transcriptswith 2′-deoxythymidine and 2′-deoxycytidine residues at templatepositions +1 and +2 vs. a transcription template with cytidine residuesat template positions +1 and +2. Sufficient quantities of mRNA to bedetected by liquid chromatography mass spectroscopy (LC-MS) weresubjected to a capping assay. In this assay, a small fragment wascleaved from the 5′ end of a full length mRNA and analyzed by LC-MS.Prior to cleavage of the mRNA, it was treated with Antarctic phosphatase(New England Biolabs catalog # M0289) to convert uncappedmonophosphates, diphosphates and triphosphates to a 5′ OH to facilitateanalysis. The phosphatase treated mRNA was then cleaved and purified.The purified RNA was subjected to LC-MS analysis. FIG. 13 shows the LCtraces. LC peaks corresponding to uncapped and Cap 1 are indicated withobserved masses. The inset schematic shows the alignment of theinitiating oligonucleotide primer on the transcription template.Subscript “m” indicates a 2′-O-methyl group and superscript “m7”indicates a base methylation. FIGS. 13A and 13B transcripts weretranscribed from templates with 2′-deoxythymidine and 2′-deoxycytidineat template nucleotides +1 and +2, respectively, using Primer/NTPFormulations 2 and 3, respectively. FIGS. 13C and 13D were transcriptstranscribed from templates with 2′-deoxycytidines at templatenucleotides +1 and +2 using Primer/NTP Formulations 2 and 3,respectively. When the ^(m7)GpppA_(2′Ome)pG initiating oligonucleotidewas completely complementary to the +1 and +2 nucleotides, the majorproduct observed was the desired templated Cap 1 transcript initiatedwith ^(m7)GpppA_(2′Ome)pG . . . (FIGS. 13A and 13B). For FIGS. 13A and13B, an estimate of the capping efficiency was made using the followingformula (intensity of capped peaks)/[(intensity of cappedpeaks)+(intensity of the 5′ OH peak)] and capping efficiency withPrimer/NTP Formulation 2 and Primer/NTP Formulation 3 was 99% and 96%,respectively. Only minor aberrant initiation products were detected. Incontrast, when template nucleotides +1 and +2 are cytidine, there is notperfect complementarity between the ^(m7)GpppA_(2′Ome)pG initiatingoligonucleotide and these template nucleotides. FIGS. 13C and 13D showthat the initiating capped oligonucleotide initiated in two registers.In the first register, the 3′ guanosine initiating cappedoligonucleotide residue paired with the +1 template cytidine to producea transcript with a an additional untemplated 5′ adenosine (Cap 1+A).Note that “|” indicates a base pair of the capped initiating nucleotidewith the template nucleotide in the schematic. Designated templatenucleotide position is indicated. In the second register, the 3′guanosine initiating capped oligonucleotide residue paired with the +2template cytidine and the +1 initiating capped oligonucleotide adenosinedoes not form a complete hybrid with the +1 template nucleotide. Thisproduces a transcript where the templated 5′ guanosine has been replacedwith an untemplated adenosine (Cap 1 GtoA). An estimate of the cappingefficiency was made using the following formula (intensity of cappedpeaks “Cap1+A”+“Cap1 GtoA”)/[(intensity of capped peaks “Cap 1+A”+“Cap 1GtoA”)+(intensity of the 5′ OH peak)]. The calculated capping efficiencyin FIGS. 13C and 13D were 97 and 77%. We note the surprising findingthat capping efficiency and templated transcription initiation fidelitywere much higher when the initiating capped oligonucleotide primer wascompletely complementary to the corresponding template +1 and +2nucleotides. In addition, with our method, efficient capping can beachieved without reducing the concentration of an NTP to drive capping,allowing much greater transcription yields. Thus the capping method wedescribe is distinct from and superior to that of Ishikawa et al.

Example 19 FIG. 14A-14B Comparison of Translation in DifferentiatedTHP-1 Cells of mRNA Made with ^(m7)GpppA_(2′Ome)pG Initiating CappedOligonucleotide on a Transcription Template with 2′-deoxythymidine and2′-deoxycytidine Residues at Template Positions +1 and +2 Vs. aTranscription Template with Cytidine Residues at Template Positions +1and +2

In order to assess the expression of luciferase mRNAs generated inExamples 14 and 15, mRNAs were transfected into THP-1 cells (ATCC,Catalog# TIB-202) with six replicates. THP-1 cells were cultured in ATCCformulated RPMI-1640 (ATCC, Catalog#30-2001) supplemented with 10% FBS,sodium pyruvate and penicillin/streptomycin at 37° C. under anatmosphere of 5% CO2. Cells were seeded at 2E+05 cells per well in a24-well plate format in the presence of the phorbol ester12-O-Tetradecanoylphorbol-13-acetate (TPA; Cell Signaling Technologies,Catalog#4174) to induce differentiation. Cells were transfected with 100ng of mRNA per well 72 hours after seeding. For comparison, cells werealso transfected with a Cap0 luciferase mRNA generated by initiationwith ARCA. At 20 hours post-transfection, cells were harvested andluciferase activity was measured using a ONE-Glo Luciferase Assay Systemkit (Promega Catalog# E6120) according to the manufacturer'srecommendations. Luminescence was measured using a GloMax-Multi+Detection System instrument according to the manufacturer'srecommendations. Data were graphed as the mean of six replicates+/−thestandard deviation from the mean. Data were analyzed using the Unpairedt-test to generate p values as a measure of significance. The p-value isbetween pairs is indicated. Translation was compared in THP-1 cells fortranscripts were generated with ^(m7)GpppA_(2′Ome)pG initiating cappedoligonucleotide and a template comprising 2′-deoxythymidine and2′-deoxycytidine residues at template positions +1 and +2 (“TC”Template) vs. a transcription template With cytidine residues attemplate Positions +1 and +2 (“CC” Template). A) Transcription wasconducted with Primer/NTP Formulations 2 or B) Primer/NTP Formulations3. With both formulations, translation in cultured THP-1 cells wassignificantly superior when the ^(m7)GpppA_(2′Ome)pG initiating cappedoligonucleotide primer was completely complementary to templatenucleotides +1 and +2 (“TC” template) as described in the currentinvention. In FIG. 14B, we also assessed the activity of an mRNA thatwas made with the “TC” Template and capped with ARCA (Cap 0). This isthe current industry standard for generating co-transcriptionally cappedmRNAs. The ARCA capped RNA had significantly less activity thantranscripts made where the ^(m7)GpppA_(2′Ome)pG initiating cappedoligonucleotide primer was completely complementary to templatenucleotides +1 and +2 (“TC” template).

In sum, the Examples shown here demonstrate that, relative to previouslypublished methods, the methods described here generate RNAs with acombination of 1) high yield, 2) high extent of capping, 3) highfidelity of templated transcription and 4) superior activity in cells.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms “a” and“an” and “the” and similar referents in the context of describing theinvention (especially in the context of the following claims) are to beconstrued to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. The terms“comprising”, “having,” “including,” containing”, etc. shall be readexpansively and without limitation (e.g., meaning “including, but notlimited to,”). Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed with reference tocertain embodiments and optional features, modification and variation ofthe inventions embodied therein herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention. Thus, it should beunderstood that although the present invention has been specificallydisclosed by reference to certain embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of certain embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A complex comprising an initiating cappedoligonucleotide primer and a DNA template, wherein the initiating cappedoligonucleotide primer comprises a structure:

wherein B₁ and B₂ are independently a natural, a modified, or anunnatural nucleoside base; and R₁, R₂, and R₃ are independently OH orO-methyl; wherein the DNA template comprises a promoter regioncomprising a transcriptional start site having a first nucleotide atnucleotide position +1 and a second nucleotide at nucleotide position+2; and wherein the initiating capped oligonucleotide primer ishybridized to the DNA template at least at nucleotide positions +1 and+2.
 2. The complex of claim 1, wherein B₁ is at nucleotide position +1and B₂ is at nucleotide position +2.
 3. The complex of claim 1, whereinB₁ and B₂ are natural nucleoside bases.
 4. The complex of claim 1,wherein at least one of B₁ and B₂ is a modified or an unnatural base. 5.The complex of claim 1, wherein at least one of B₁ and B₂ isN6-methyladenine.
 6. The complex of claim 1, wherein B₁ is adenine andB₂ is guanine.
 7. The complex of claim 1, wherein B₁ is guanine and B₂is guanine.
 8. The complex of claim 1, wherein B₁ is adenine and B₂ isuracil.
 9. The complex of claim 1, wherein R₃ is OH.
 10. The complex ofclaim 1, wherein R₃ is O-methyl.
 11. The complex of claim 1, wherein atleast one of R₁ and R₂ is O-methyl, B₁ is adenine, and B₂ is guanine.12. The complex of claim 1, wherein at least one of R₁ and R₂ isO-methyl, and B₁ and B₂ are guanine.
 13. The complex of claim 1, whereinat least one of R₁ and R₂ is O-methyl, B₁ is adenine, and B₂ is uracil.14. The complex of claim 1, wherein B₁ is N6-methyladenine and B₂ is anatural, a modified, or an unnatural base.
 15. The complex of claim 1,wherein the initiating capped oligonucleotide primer comprises thestructure:


16. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


17. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


18. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


19. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


20. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


21. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


22. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


23. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


24. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


25. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


26. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


27. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


28. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


29. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


30. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure:


31. The complex of claim 1, wherein the initiating cappedoligonucleotide primer comprises the structure: